Reprogramming progenitor compositions and methods of use thereof

ABSTRACT

The invention generally features compositions comprising induced pluripotent stem cell progenitors (also termed reprogramming progenitor cells) and methods of isolating such cells. The invention also provides compositions comprising induced pluripotent stem cells (iPSCs) derived from such progenitor cells. Induced pluripotent stem cell progenitors generate iPSCs at high efficiency.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application U.S. Ser. No.15/552,476, filed on Aug. 21, 2017, which is a U.S. national stageapplication, pursuant to 35 U.S.C. § 371, of International PCTApplication No. PCT/US2016/019911, filed on Feb. 26, 2016, designatingthe United States and published in English, which claims the benefit ofand priority to U.S. Provisional Application Ser. No. 62/126,417, filedon Feb. 27, 2015, the contents of all of which are incorporated hereinby reference in their entireties.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with US government support under HD105278,DK057978, DK062434, and DK063491 awarded by the National Institutes ofHealth. The US government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on Nov. 3, 2020, isnamed 167776 010407US_SL.txt and is 210,140 bytes in size.

BACKGROUND OF THE INVENTION

A need exists for cell-based compositions to repair or replace damagedor diseased tissues or organs. In the United States alone, thousands ofpatients die every year waiting for donor organs to become availablebecause the need for transplantable organs far exceeds the supply. Inaddition, many serious medical conditions, such as neurodegenerativedisorders, heart disease, and diabetes, could be helped by cell-basedtherapies. One limitation to the development of cell-based therapies isthe lack of a reliable source of pluripotent stem cells.

SUMMARY OF THE INVENTION

As described below, the invention generally features compositionscomprising induced pluripotent stem cell progenitors (also termedreprogramming progenitor cells) and methods of isolating such cells. Theinvention also provides compositions comprising induced pluripotent stemcells (iPSCs) derived from such progenitor cells. Induced pluripotentstem cell progenitors generate iPSCs at high efficiency.

In one aspect, the invention provides a method for selecting a mammalianinduced pluripotent stem cell progenitor, the method involving isolatingan induced pluripotent stem cell progenitor expressing one or more ofOct4, Sox2, Klf4 and cMyc, and having increased expression of anestrogen related receptor relative to a reference cell, therebyselecting an induced pluripotent stem cell progenitor.

In another aspect, the invention provides a method for selecting amammalian induced pluripotent stem cell progenitor, the method involvingisolating an induced pluripotent stem cell progenitor expressing one ormore of Oct4, Sox2, Klf4 and cMyc, having reduced expression of Sca1 andCD34, and having increased expression of an estrogen related receptorrelative to a reference cell, thereby selecting an induced pluripotentstem cell progenitor.

In yet another aspect, the invention provides a method of isolating acell population enriched for induced pluripotent stem cell progenitors,the method involving isolating one or more induced pluripotent stem cellprogenitors expressing Oct4, Sox2, Klf4 and cMyc, and having increasedexpression of an estrogen related receptor relative to a reference cell,and culturing the one or more mammalian induced pluripotent stem cellprogenitors to obtain a cell population enriched for induced pluripotentstem cell progenitors.

In still another aspect, the invention provides a method of obtaining amurine induced pluripotent stem cell progenitor, the method involvingexpressing Oct4, Sox2, Klf4 and cMyc in a murine cell in culture,isolating from the culture a cell having reduced expression of Sca1 andCD34 and having increased expression of ERRγ relative to a referencecell, and culturing the cell to obtain an induced pluripotent stem cellprogenitor. In one embodiment, the murine cell is a mouse embryonicfibroblast. In another embodiment, the cell further expresses anincreased level of PGC-1β and/or IDH3 relative to a reference cell.

In another aspect, the invention provides a method of obtaining a humaninduced pluripotent stem cell progenitor, the method involvingexpressing Oct4, Sox2, Klf4 and cMyc in a human cell in culture,isolating from the culture a cell having increased expression of ERRαand/or PGC-1α and/or PGC-1β and/or IDH3 relative to a reference cell,thereby obtaining a human induced pluripotent stem cell progenitor.

In yet another aspect, the invention provides an induced pluripotentstem cell progenitor obtained according to the above aspects or anyother aspect of the invention delineated herein or various embodimentsof the above aspects or any other aspect of the invention delineatedherein.

In still another aspect, the invention provides a method for generatinga induced pluripotent stem cell progenitor or induced pluripotent stemcell, the method involving expressing recombinant estrogen relatedreceptor (ERR) alpha or gamma in a cell expressing Oct4, Sox2, Klf4 andcMyc and culturing the cell, thereby generating a induced pluripotentstem cell progenitor or induced pluripotent stem cell. In oneembodiment, the cell also expresses PGC-1α, PGC-1β, and/or IDH3. Inanother embodiment, the cell is Sca1⁻CD34⁻. In yet another embodiment,the cell or cells include retroviral vectors encoding Oct4, Sox2, Klf4and cMyc.

In another aspect, the invention provides a cellular compositioncontaining an effective amount of an induced pluripotent stem cell orcellular descendant thereof in a pharmaceutically acceptable excipient.In one embodiment, the induced pluripotent stem cell is capable ofgiving rise to a pancreatic cell, neuronal cell, or cardiac cell.

In yet another aspect, the invention provides a kit containing aninduced pluripotent stem cell or progenitor thereof obtained accordingto the above aspects or any other aspect of the invention delineatedherein or various embodiments of the above aspects or any other aspectof the invention delineated herein.

In still another aspect, the invention provides an expression vectorcontaining a promoter sequence of an oxidative or glycolytic pathwaygene operably linked to a polynucleotide encoding a detectablepolypeptide. In one embodiment, the promoter is sufficient to direct orenhance transcription of an ERR polynucleotide. In another embodiment,the vector is a lentiviral vector. In yet another embodiment, thepromoter comprises an ERR alpha enhancer sequence. In still anotherembodiment, the promoter comprises at least about nucleotide positions64072402-64073375 of chromosome 11.

In another aspect, the invention provides a mammalian cell containingthe expression vector containing a promoter sequence of an oxidative orglycolytic pathway gene operably linked to a polynucleotide encoding adetectable polypeptide. In one embodiment, the cell further contains apolynucleotide sequence encoding one or more of Oct4, Sox2, Klf4 andcMyc.

In yet another aspect, the invention provides a method of selecting acell having increased oxidative and/or glycolytic pathway activity, themethod involving detecting an increase in the level or activity of aprotein or polynucleotide listed in FIG. 7. In one embodiment, the cellcontains an expression vector containing a polynucleotide sequence thatis 5′ of the open reading frame encoding said protein and that directsexpression of said open reading frame. In another embodiment, the cellcontains an expression vector containing a polynucleotide encoding aprotein listed in FIG. 7 fused to a detectable polypeptide. In yetanother embodiment, the detectable polypeptide is selected from thegroup consisting of GFP, RFP, YFP, and luciferase.

In still another aspect, the invention provides a method of selecting acell having increased oxidative and/or glycolytic pathway activity, themethod involving detecting an increase in levels of a reactive oxygenspecies.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the estrogen related receptor is ERRα, ERRβor ERRγ. In various embodiments of the above aspects or any other aspectof the invention delineated herein, the cell further expresses anincreased level of PGC-1 α, PGC-1β, and/or IDH3 relative to a referencecell. In various embodiments of the above aspects or any other aspect ofthe invention delineated herein, the induced pluripotent stems cellprogenitor is a human or murine cell. In various embodiments of theabove aspects or any other aspect of the invention delineated herein,the induced pluripotent stem cell progenitor is obtained by expressingOct4, Sox2, Klf4 and/or cMyc in a cell that is a fibroblast, embryonicfibroblast, human lung fibroblast, adipose stem cell, or IMR90 cell.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the induced pluripotent stem cellprogenitor expresses Oct4, Sox2, Klf4 and cMyc. In various embodimentsof the above aspects or any other aspect of the invention delineatedherein, the reference cell expresses Sca 1 and/or CD34 or a humanortholog or functional equivalent thereof. In various embodiments of theabove aspects or any other aspect of the invention delineated herein,the reference cell fails to express detectable levels of one or more ofOct4, Sox2, Klf4 and cMyc. In various embodiments of the above aspectsor any other aspect of the invention delineated herein, the cell orcells express undetectable levels of Sca1 and CD34 proteins or humanorthologs thereof, or polynucleotides encoding said proteins. In variousembodiments of the above aspects or any other aspect of the inventiondelineated herein, the cell or cells display an increased metabolic ratedefined by increased extracellular acidification rate and/or oxygenconsumption rate relative to a reference cell. In various embodiments ofthe above aspects or any other aspect of the invention delineatedherein, ERRγ and/or PGC-1β expression is at least about 2, 5 or 10 foldhigher than the level in a reference cell. In various embodiments of theabove aspects or any other aspect of the invention delineated herein,polynucleotide expression level is determined by qPCR analysis. Invarious embodiments of the above aspects or any other aspect of theinvention delineated herein, the cell or cells contains one or moreretroviral vectors encoding Oct4, Sox2, Klf4 and cMyc. In variousembodiments of the above aspects or any other aspect of the inventiondelineated herein, the induced pluripotent stem cells arehyper-energetic cells.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the cell or cells has increased in one ormore of nicotinamide adenine dinucleotide (NADH), α-ketoglutarate,cellular ATP, NADH/NAD+ ratio, ATP synthase in mitochondria (ATP5G1),succinate dehydrogenase (SDHB), isocitrate dehydrogenase (IDH3) and NADHdehydrogenase (NDUFA2), superoxide dismutase 2 (SOD2), NADPH oxidase 4(NOX4) and catalase (CAT) were increased about five days followingexpression of Oct4, Sox2, Klf4 and cMyc. In various embodiments of theabove aspects or any other aspect of the invention delineated herein,the cell or cells has increased gene expression profile or activity inone or more pathways listed in FIG. 10B. In various embodiments of theabove aspects or any other aspect of the invention delineated herein,the cell or cells has a decreased methylation level of an amino acid ofa histone in a promoter or an enhancer region associated with genes thatfunction in fibroblast identity relative to a reference cell. In variousembodiments of the above aspects or any other aspect of the inventiondelineated herein, the cell or cells has an increased methylation levelof an amino acid of a histone in a promoter or an enhancer regionassociated with genes that function in reprogramming relative to areference cell. In various embodiments of the above aspects or any otheraspect of the invention delineated herein, the reference cell does notexpress detactable ERRα. In various embodiments of the above aspects orany other aspect of the invention delineated herein, the histone is H3histone, and the amino acid is a lysine located at fourth (4th) aminoacid position from a N-terminal of the histone.

Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “induced pluripotent stem cell progenitor” also termed a“reprogramming progenitor” is meant a cell that gives rise to an inducedpluripotent stem cell.

By “Sca1 polypeptide” is meant a protein or fragment thereof having atleast 85% amino acid sequence identity to the sequence provided at NCBIRef: NP_001258375.1 and having SCA1 antigenicity. An exemplary murineamino acid sequence is provided below:

(SEQ ID NO: 1) MDTSHTTKSCLLILLVALLCAERAQGLECYQCYGVPFETSCPSITCPYPDGVCVTQEAAVIVDSQTRKVKNNLCLPICPPNIESMEILGTKVNVKTSCCQEDLCNVAVPNGGSTWTMAGVLLFSLSSVLLQTLL

By “Sca1 polynucleotide” is meant any nucleic acid molecule encoding aSca1 polypeptide or fragment thereof. An exemplary murine Sca1 nucleicacid sequence is provided at NCBI Ref NM_001271446.1, and reproducedbelow:

(SEQ ID NO: 2) 1 cttaaccaat aaacatgatg gcctggaaaa ggttaagtactgaaacccct ccctcttcag 61 gatgccagct gggaggagct gaaggaaatt aaagtacttcagtccacatc tgacagaact 121 tgccactgtg cctgcaacct tgtctgagag gaagtaaggactggtgtgag gagggagctc 181 ccttctctga ggatggacac ttctcacact acaaagtcctgtttgctgat tcttcttgtg 241 gccctactgt gtgcagaaag agctcaggga ctggagtgttaccagtgcta tggagtccca 301 tttgagactt cttgcccatc aattacctgc ccctaccctgatggagtctg tgttactcag 361 gaggcagcag ttattgtgga ttctcaaaca aggaaagtaaagaacaatct ttgcttaccc 421 atctgccctc ctaatattga aagtatggag atcctgggtactaaggtcaa cgtgaagact 481 tcctgttgcc aggaagacct ctgcaatgta gcagttcccaatggaggcag cacctggacc 541 atggcagggg tgcttctgtt cagcctgagc tcagtcctcctgcagacctt gctctgatgg 601 tcctcccaat gacctccacc cttgtccttt tatcctcatgtgcaacaatt cttcctggag 661 ccctctagtg atgaattatg agttatagaa gctccaaggtgggagtagtg tgtgaaatac 721 catgttttgc ctttatagcc cctgctgggt aggtaggtgctctaatcctc tctagggctt 781 tcaagtctgt acttcctaga atgtcatttt gttgtggattgctgctcatg accctggagg 841 cacacagcca gcacagtgaa gaggcagaat tccaaggtattatgctatca ccatccacac 901 ataagtatct ggggtcctgc aatgttccca catgtatcctgaatgtcccc ctgttgagtc 961 caataaaccc tttgttctcc ca

By “CD34 polypeptide” is meant a protein or fragment thereof having atleast 85% homology to the sequence provided at NCBI Ref: NP_001020280.1(human) or NCBI Ref: NP_001104529.1 (murine).

An exemplary human amino acid sequence is provided below:

(SEQ ID NO: 3) 1 mlvrrgarag prmprgwtal cllsllpsgf msldnngtatpelptqgtfs nvstnvsyqe 61 tttpstlgst slhpvsqhgn eattnitett vkftstsvitsvygntnssv gsgtsvistv 121 fttpanvstp ettlkpslsp gnvsdlstts tslatsptkpytssspilsd ikaeikcsgi 181 revkltqgic leqnktssca efkkdrgegl arvlogeeqadadagaqvcs lllagsevrp 241 qclllvlanr teissklqlm kkhqsdlkkl gildfteqdvashqsysqkt lialvtsgal 301 lavlgitgyf lmnrrswspt gerlgedpyy tengggqgyssgpgtspeaq gkasvnrgaq 361 engtgqatsr nghsarqhvv adtel

An exemplary murine amino acid sequence is provided below:

(SEQ ID NO: 4) MQVHRDTRAGLLLPWRWVALCLMSLLHLNNLTSATTETSTQGISPSVPTNESVEENITSSIPGSTSHYLIYQDSSKTTPAISETMVNFTVTSGIPSGSGTPHTESQPQTSPTGILPTTSDSISTSEMTWKSSLPSINVSDYSPNNSSFEMTSPTEPYAYTSSSAPSAIKGEIKCSGIREVRLAQGICLELSEASSCEEFKKEKGEDLIQILCEKEEAEADAGASVCSLLLAQSEVRPECLLMVLANSTELPSKLQLMEKHQSDLRKLGIQSFNKQDIGSHQSYSRKTLIALVTSGVLLAILGTTGY FLMNRRSWSPTGERLELEP 

By “CD34 polynucleotide” is meant any nucleic acid sequence encoding anCD34 polypeptide or fragment thereof.

An exemplary human CD34 nucleic acid sequence is provided at NCBI RefNM_001025109.1:

(SEQ ID NO: 5) 1 ccttttttgg cctcgacggc ggcaacccag cctccctcctaacgccctcc gcctttggga 61 ccaaccaggg gagctcaagt tagtagcagc caaggagaggcgctgccttg ccaagactaa 121 aaagggaggg gagaagagag gaaaaaagca agaatcccccacccctctcc cgggcggagg 181 gggcgggaag agcgcgtcct ggccaagccg agtagtgtcttccactcggt gcgtctctct 241 aggagccgcg cgggaaggat gctggtccgc aggggcgcgcgcgcagggcc caggatgccg 301 cggggctgga ccgcgctttg cttgctgagt ttgctgccttctgggttcat gagtcttgac 361 aacaacggta ctgctacccc agagttacct acccagggaacattttcaaa tgtttctaca 421 aatgtatcct accaagaaac tacaacacct agtacccttggaagtaccag cctgcaccct 481 gtgtctcaac atggcaatga ggccacaaca aacatcacagaaacgacagt caaattcaca 541 tctacctctg tgataacctc agtttatgga aacacaaactcttctgtcca gtcacagacc 601 tctgtaatca gcacagtgtt caccacccca gccaacgtttcaactccaga gacaaccttg 661 aagcctagcc tgtcacctgg aaatgtttca gacctttcaaccactagcac tagccttgca 721 acatctccca ctaaacccta tacatcatct tctcctatcctaagtgacat caaggcagaa 781 atcaaatgtt caggcatcag agaagtgaaa ttgactcagggcatctgcct ggagcaaaat 841 aagacctcca gctgtgcgga gtttaagaag gacaggggagagggcctggc ccgagtgctg 901 tgtggggagg agcaggctga tgctgatgct ggggcccaggtatgctccct gctccttgcc 961 cagtctgagg tgaggcctca gtgtctactg ctggtcttggccaacagaac agaaatttcc 1021 agcaaactcc aacttatgaa aaagcaccaa tctgacctgaaaaagctggg gatcctagat 1081 ttcactgagc aagatgttgc aagccaccag agctattcccaaaagaccct gattgcactg 1141 gtcacctcgg gagccctgct ggctgtcttg ggcatcactggctatttcct gatgaatcgc 1201 cgcagctgga gccccacagg agaaaggctg ggcgaagacccttattacac ggaaaacggt 1261 ggaggccagg gctatagctc aggacctggg acctcccctgaggctcaggg aaaggccagt 1321 gtgaaccgag gggctcagga aaacgggacc ggccaggccacctccagaaa cggccattca 1381 gcaagacaac acgtggtggc tgataccgaa ttgtgactcggctaggtggg gcaaggctgg 1441 gcagtgtccg agagagcacc cctctctgca tctgaccacgtgctaccccc atgctggagg 1501 tgacatctct tacgcccaac ccttccccac tgcacacacctcagaggctg ttcttggggc 1561 cctacacctt gaggaggggc aggtaaactc ctgtcctttacacattcggc tccctggagc 1621 cagactctgg tcttctttgg gtaaacgtgt gacgggggaaagccaaggtc tggagaagct 1681 cccaggaaca atcgatggcc ttgcagcact cacacaggacccccttcccc taccccctcc 1741 tctctgccgc aatacaggaa cccccagggg aaagatgagcttttctaggc tacaattttc 1801 tcccaggaag ctttgatttt taccgtttct tccctgtattttctttctct actttgagga 1861 aaccaaagta accttttgca cctgctctct tgtaatgatatagccagaaa aacgtgttgc 1921 cttgaaccac ttccctcatc tctcctccaa gacactgtggacttggtcac cagctcctcc 1981 cttgttctct aagttccact gagctccatg tgccccctctaccatttgca gagtcctgca 2041 cagttttctg gctggagcct agaacaggcc tcccaagttttaggacaaac agctcagttc 2101 tagtctctct ggggccacac agaaactctt tttgggctcctttttctccc tctggatcaa 2161 agtaggcagg accatgggac caggtcttgg agctgagcctctcacctgta ctcttccgaa 2221 aaatcctctt cctctgaggc tggatcctag ccttatcctctgatctccat ggcttcctcc 2281 tccctcctgc cgactcctgg gttgagctgt tgcctcagtcccccaacaga tgcttttctg 2341 tctctgcctc cctcaccctg agccccttcc ttgctctgcacccccatatg gtcatagccc 2401 agatcagctc ctaaccctta tcaccagctg cctcttctgtgggtgaccca ggtccttgtt 2461 tgctgttgat ttctttccag aggggttgag cagggatcctggtttcaatg acggttggaa 2521 atagaaattt ccagagaaga gagtattggg tagatattttttctgaatac aaagtgatgt 2581 gtttaaatac tgcaattaaa gtgatactga aacacaaaaa a

An exemplary murine CD34 nucleic acid sequence is provided at NCBI Ref:NM_001111059.1:

(SEQ ID NO: 6) 1 ggggataagc cagcatcccc cacccactcc ggacagggagcaggggagga gagccaatat 61 cccccacccc tgcgcagggc ggaggagcgc gtcccgcgccgggccgcctc ctgcaccgag 121 cgcatctccg gagcggtaca ggagaatgca ggtccacagggacacgcgcg cggggctcct 181 gctgccatgg cgctgggtag ctctctgcct gatgagtctgctgcatctaa ataacttgac 241 ttctgctacc acggagactt ctacacaagg aatatccccatcagttccta ccaatgagtc 301 tgttgaggaa aatatcacat ctagcatccc tggaagtaccagccactact tgatctatca 361 ggacagcagt aagaccacac cagccatctc agagactatggtcaacttta cagttacctc 421 tgggatccct tcaggctctg gaactccaca cactttttcacaaccacaga cttccccaac 481 tggcatactg cctactactt cagacagtat ttccacttcagagatgacct ggaagtccag 541 cctgccatct ataaatgttt ctgattattc gcctaataatagcagctttg agatgacatc 601 acccaccgag ccatatgctt acacatcatc ttctgctccgagtgccatta agggagaaat 661 caaatgctct ggaatccgag aagtgaggtt ggcccagggtatctgcctgg aactaagtga 721 agcatctagt tgtgaggagt ttaagaagga aaagggagaagatctaattc aaatactgtg 781 tgaaaaggag gaggctgagg ctgatgctgg tgctagtgtctgctccctgc ttctagccca 841 gtctgaggtt aggcctgagt gtttgctgat ggtcttggccaatagcacag aacttcccag 901 caaactccag cttatggaaa agcaccaatc tgacttgagaaagctgggga tccaaagctt 961 caataaacaa gatatcggga gccaccagag ctattcccgaaagactctta ttgcattggt 1021 cacctctgga gttctgctgg ccatcttggg caccactggttatttcctga tgaaccgtcg 1081 cagttggagc cctacaggag aaaggctgga gctggaaccttgatggctgt tgggaagaaa 1141 agaggctgca catgtagctg tacctgctct gccccccccccactcctact tcctttgtgc 1201 tctcctcaca gtacctcaca accctgctta ccagataatgctactttatt tctatactgt 1261 ccagggtgaa gacccttatt acacggagaa tggtggaggccagggctata gctcaggacc 1321 tggggcctcc cctgagactc agggaaaggc caatgtgacccgaggggctc aggagaacgg 1381 gaccggccag gccacttcca gaaacggcca ttcagcaagacaacatgtgg tggctgacac 1441 agaactgtga tttggttggg tgggcaactg ggtggtatgcaggaaagtgg catctcttgt 1501 ctctgacttc atgctgcctt cagctcatgt ccggccttctcctattacat acacttctga 1561 aactgttcct gggactcttc accttgggga aggcagataaactgccttct gcacattcaa 1621 cttcctgaat ccaatctctg acctttgggt caagttgtggtgggaagaag cctaggtcta 1681 gaggagctgc caaaaaagtt ggtggctatg tagcacttgccctggaccca tttctcctct 1741 ctcgcctctt cacgggaact ctccggaaga ctagcttttctaagctacca cttcttccca 1801 ggaaactttg ctatttttac tgcttcttcc cctactttatggaaaccaag gtattcactg 1861 acatgtgctc ccttgcaagg gtacagccag aaaagtgctattttaaaata catccttaaa 1921 aaatgcatcc cttataactt caagacactg tggatttagtcaccaacttc tatcttgttc 1981 acctgttcct gaatgtctgt ctacagaggc caggacaactttctgtctgg agtctgctca 2041 atgttttaga gcaacagctc aatctgatcc cttgggcccacacagaaatc tcattggttc 2101 aacctagaca ggacagtgga attagacttt gaactgagcctctgtttttt gttttatttt 2161 attgctgggg tttgaaccca gagcttcaca cagcttctttaggcttccaa gtagcttgag 2221 ctaccaggcc cagctgagct aaacctcctg acctgagctcttcaaaggaa tactcttgct 2281 ctgaggccct tggccttctc taaattacgt gacttcccccttcctctgac tcctggggga 2341 gctgtggcct cagtcccctg gcagattcct ttcagtctgtgcctttccta gtccaaaccc 2401 cttcactatt ttataaccct ttgtgatcag aggttcagaatatctacaaa gactataagc 2461 ttcctctcct ggggttaagg ggagaacagg ggtcctgattttaatgatgg ctaggaacaa 2521 aactttccag agatgagagg attgggtgta ttctcttctgaataaacgtg atgagtgaaa 2581 atgatgtaat taaattgatg atgaaatatt tgatgtggcc c

By “cMyc polypeptide” is meant a protein or fragment thereof having atleast 85% homology to the sequence provided at NCBI Ref: NP_002458.2(human) or NP_001170823.1 (murine). An exemplary human amino acidsequence is provided below:

(SEQ ID NO: 7) MDFFRVVENQQPPATMPLNVSETNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGESAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVEPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRNSCAAn exemplary murine amino acid sequence is provided below:

(SEQ ID NO: 8) MPLNVNETNRNYDLDYDSVQPYFICDEEENFYHQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVATSFSPREDDDGGGGNESTADQLEMMTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGESAAAKLVSEKLASYQAARKDSTSLSPARGHSVCSTSSLYLQDLTAAASECIDPSVVEPYPLNDSSSPKSCTSSDSTAFSPSSDSLLSSESSPRASPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQTPAKRSESGSSPSRGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRAKLDSGRVLKQISNNRKCSSPRSSDTEENDKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSIQADEHKLTSEKDLLRKRREQLKHKLEQLRNSGA

By “cMyc” is meant a nucleic acid molecule encoding a cMyc polypeptide.An exemplary human cMyc polynucleotide sequence is provided atNM_002467.4, the sequence of which is reproduced below:

(SEQ ID NO: 9) 1 gacccccgag ctgtgctgct cgcggccgcc accgccgggccccggccgtc cctggctccc 61 ctcctgcctc gagaagggca gggcttctca gaggcttggcgggaaaaaga acggagggag 121 ggatcgcgct gagtataaaa gccggttttc ggggctttatctaactcgct gtagtaattc 181 cagcgagagg cagagggagc gagcgggcgg ccggctagggtggaagagcc gggcgagcag 241 agctgcgctg cgggcgtcct gggaagggag atccggagcgaatagggggc ttcgcctctg 301 gcccagccct cccgctgatc ccccagccag cggtccgcaacccttgccgc atccacgaaa 361 ctttgcccat agcagcgggc gggcactttg cactggaacttacaacaccc gagcaaggac 421 gcgactctcc cgacgcgggg aggctattct gcccatttggggacacttcc ccgccgctgc 481 caggacccgc ttctctgaaa ggctctcctt gcagctgcttagacgctgga tttttttcgg 541 gtagtggaaa accagcagcc tcccgcgacg atgcccctcaacgttagctt caccaacagg 601 aactatgacc tcgactacga ctcggtgcag ccgtatttctactgcgacga ggaggagaac 661 ttctaccagc agcagcagca gagcgagctg cagcccccggcgcccagcga ggatatctgg 721 aagaaattcg agctgctgcc caccccgccc ctgtcccctagccgccgctc cgggctctgc 781 tcgccctcct acgttgcggt cacacccttc tcccttcggggagacaacga cggcggtggc 841 gggagcttct ccacggccga ccagctggag atggtgaccgagctgctggg aggagacatg 901 gtgaaccaga gtttcatctg cgacccggac gacgagaccttcatcaaaaa catcatcatc 961 caggactgta tgtggagcgg cttctcggcc gccgccaagctcgtctcaga gaagctggcc 1021 tcctaccagg ctgcgcgcaa agacagcggc agcccgaaccccgcccgcgg ccacagcgtc 1081 tgctccacct ccagcttgta cctgcaggat ctgagcgccgccgcctcaga gtgcatcgac 1141 ccctcggtgg tcttccccta ccctctcaac gacagcagctcgcccaagtc ctgcgcctcg 1201 caagactcca gcgccttctc tccgtcctcg gattctctgctctcctcgac ggagtcctcc 1261 ccgcagggca gccccgagcc cctggtgctc catgaggagacaccgcccac caccagcagc 1321 gactctgagg aggaacaaga agatgaggaa gaaatcgatgttgtttctgt ggaaaagagg 1381 caggctcctg gcaaaaggtc agagtctgga tcaccttctgctggaggcca cagcaaacct 1441 cctcacagcc cactggtcct caagaggtgc cacgtctccacacatcagca caactacgca 1501 gcgcctccct ccactcggaa ggactatcct gctgccaagagggtcaagtt ggacagtgtc 1561 agagtcctga gacagatcag caacaaccga aaatgcaccagccccaggtc ctcggacacc 1621 gaggagaatg tcaagaggcg aacacacaac gtcttggagcgccagaggag gaacgagcta 1681 aaacggagct tttttgccct gcgtgaccag atcccggagttggaaaacaa tgaaaaggcc 1741 cccaaggtag ttatccttaa aaaagccaca gcatacatcctgtccgtcca agcagaggag 1801 caaaagctca tttctgaaga ggacttgttg cggaaacgacgagaacagtt gaaacacaaa 1861 cttgaacagc tacggaactc ttgtgcgtaa ggaaaagtaaggaaaacgat tccttctaac 1921 agaaatgtcc tgagcaatca cctatgaact tgtttcaaatgcatgatcaa atgcaacctc 1981 acaaccttgg ctgagtcttg agactgaaag atttagccataatgtaaact gcctcaaatt 2041 ggactttggg cataaaagaa cttttttatg cttaccatcttttttttttc tttaacagat 2101 ttgtatttaa gaattgtttt taaaaaattt taagatttacacaatgtttc tctgtaaata 2161 ttgccattaa atgtaaataa ctttaataaa acgtttatagcagttacaca gaatttcaat 2221 cctagtatat agtacctagt attataggta ctataaaccctaattttttt tatttaagta 2281 cattttgctt tttaaagttg atttttttct attgtttttagaaaaaataa aataactggc 2341 aaatatatca ttgagccaaa tcttaaaaaa aaaaaaaaa

An exemplary murine cMyc polynucleotide sequence is provided atNM_001177352.1, the sequence of which is reproduced below:

(SEQ ID NO: 10) 1 cccgcccacc cgccctttat attccggggg tctgcgcggccgaggacccc tgggctgcgc 61 tgctctcagc tgccgggtcc gactcgcctc actcagctcccctcctgcct cctgaagggc 121 agggcttcgc cgacgcttgg cgggaaaaag aagggaggggagggatcctg agtcgcagta 181 taaaagaagc ttttcgggcg tttttttctg actcgctgtagtaattccag cgagagacag 241 agggagtgag cggacggttg gaagagccgt gtgtgcagagccgcgctccg gggcgaccta 301 agaaggcagc tctggagtga gaggggcttt gcctccgagcctgccgccca ctctccccaa 361 ccctgcgact gacccaacat cagcggccgc aaccctcgccgccgctggga aactttgccc 421 attgcagcgg gcagacactt ctcactggaa cttacaatctgcgagccagg acaggactcc 481 ccaggctccg gggagggaat ttttgtctat ttggggacagtgttctctgc ctctgcccgc 541 gatcagctct cctgaaaaga gctcctcgag ctgtttgaaggctggatttc ctttgggcgt 601 tggaaacccc gcagacagcc acgacgatgc ccctcaacgtgaacttcacc aacaggaact 661 atgacctcga ctacgactcc gtacagccct atttcatctgcgacgaggaa gagaatttct 721 atcaccagca acagcagagc gagctgcagc cgcccgcgcccagtgaggat atctggaaga 781 aattcgagct gcttcccacc ccgcccctgt ccccgagccgccgctccggg ctctgctctc 841 catcctatgt tgcggtcgct acgtccttct ccccaagggaagacgatgac ggcggcggtg 901 gcaacttctc caccgccgat cagctggaga tgatgaccgagttacttgga ggagacatgg 961 tgaaccagag cttcatctgc gatcctgacg acgagaccttcatcaagaac atcatcatcc 1021 aggactgtat gtggagcggt ttctcagccg ctgccaagctggtctcggag aagctggcct 1081 cctaccaggc tgcgcgcaaa gacagcacca gcctgagccccgcccgcggg cacagcgtct 1141 gctccacctc cagcctgtac ctgcaggacc tcaccgccgccgcgtccgag tgcattgacc 1201 cctcagtggt ctttccctac ccgctcaacg acagcagctcgcccaaatcc tgtacctcgt 1261 ccgattccac ggccttctct ccttcctcgg actcgctgctgtcctccgag tcctccccac 1321 gggccagccc tgagccccta gtgctgcatg aggagacaccgcccaccacc agcagcgact 1381 ctgaagaaga gcaagaagat gaggaagaaa ttgatgtggtgtctgtggag aagaggcaaa 1441 cccctgccaa gaggtcggag tcgggctcat ctccatcccgaggccacagc aaacctccgc 1501 acagcccact ggtcctcaag aggtgccacg tctccactcaccagcacaac tacgccgcac 1561 ccccctccac aaggaaggac tatccagctg ccaagagggccaagttggac agtggcaggg 1621 tcctgaagca gatcagcaac aaccgcaagt gctccagccccaggtcctca gacacggagg 1681 aaaacgacaa gaggcggaca cacaacgtct tggaacgtcagaggaggaac gagctgaagc 1741 gcagcttttt tgccctgcgt gaccagatcc ctgaattggaaaacaacgaa aaggccccca 1801 aggtagtgat cctcaaaaaa gccaccgcct acatcctgtccattcaagca gacgagcaca 1861 agctcacctc tgaaaaggac ttattgagga aacgacgagaacagttgaaa cacaaactcg 1921 aacagcttcg aaactctggt gcataaactg acctaactcgaggaggagct ggaatctctc 1981 gtgagagtaa ggagaacggt tccttctgac agaactgatgcgctggaatt aaaatgcatg 2041 ctcaaagcct aacctcacaa ccttggctgg ggctttgggactgtaagctt cagccataat 2101 tttaactgcc tcaaacttaa atagtataaa agaacttttttttatgcttc ccatcttttt 2161 tctttttcct tttaacagat ttgtatttaa ttgtttttttaaaaaaatct taaaatctat 2221 ccaattttcc catgtaaata gggccttgaa atgtaaataactttaataaa acgtttataa 2281 cagttacaaa agattttaag acatgtacca taattttttttatttaaaga cattttcatt 2341 tttaaagttg atttttttct attgttttta gaaaaaaataaaataattgg aaaaaatac

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

“Detect” refers to identifying the presence, absence or amount of theanalyte to be detected.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.Examples of diseases include diseases associated with a deficiency incell number. Such diseases include but are not limited toneurodegenerative disorders, heart disease, and diabetes.

By “effective amount” is meant the amount of a cell of the inventionrequired to ameliorate the symptoms of a disease relative to anuntreated patient. The effective amount of active compound(s) used topractice the present invention for therapeutic treatment of a diseasevaries depending upon the manner of administration, the age, bodyweight, and general health of the subject. Ultimately, the attendingphysician or veterinarian will decide the appropriate amount and dosageregimen. Such amount is referred to as an “effective” amount.

By “estrogen related receptor (ERR) alpha polypeptide” is meant aprotein having at least 85% amino acid sequence identity to anestrogen-related receptor alpha sequence provided at NCBI Ref No.NP_001269379 or NP_031979.2, or a fragment thereof havingtranscriptional regulatory activity.

The sequence of human ERR alpha also termed “ERR1” is provided below:

Err1_HUMAN Estrogen-related receptor alpha OS = Homo sapiens GN(SEQ ID NO: 11) mssqvvgiep lyikaepasp dspkgssete teppvalapgpaptrclpgh keeedgegag pgeqgggklv lsslpkrlclvcgdvasgyh ygvasceack affkrtiqgs ieyscpasneceitkrrrka cqacrftkcl rvgmlkegvr ldrvrggrqkykrrpevdpl pfpgpfpagp lavaggprkt aapvnalvshllvvepekly ampdpagpdg hlpavatlcd lfdreivvtiswaksipgfs slslsdqmsv lqsvwmevlv lgvaqrslplgdelafaedl vldeegaraa glgelgaall qlvrrlgalrlereeyvllk alalansdsv hiedaeaveq lrealhealleyeagragpg ggaerrragr llltlpllrq tagkvlahfy gvklegkvpm hklflemlea mmd

The sequence of a murine ERR alpha (NCBI Ref No. NP_031979.2)polypeptide also termed “ERR1” is provided below:

(SEQ ID NO: 12) MSSQVVGIEPLYIKAEPASPDSPKGSSETETEPPVTLASGPAPARCLPGHKEEEDGEGAGSGEQGSGKLVLSSLPKRLCLVCGDVASGYHYGVASCEACKAFFKRTIQGSIEYSCPASNECEITKRRRKACQACRFTKCLRVGMLKEGVRLDRVRGGRQKYKRRPEVDPLPFPGPFPAGPLAVAGGPRKTAPVNALVSHLLVVEPEKLYAMPDPASPDGHLPAVATLCDLFDREIVVTISWAKSIPGFSSLSLSDQMSVLQSVWMEVLVLGVAQRSLPLQDELAFAEDLVLDEEGARAAGLGDLGAALLQLVRRLQALRLEREEYVLLKALALANSDSVHIEDAEAVEQLREALHEALLEYEAGRAGPGGGAERRRAGRLLLTLPLLRQTAGKVLAHFYGVKLEGKVPMHKLFLEMLEAMMD

By “ERR alpha polynucleotide” is meant any nucleic acid sequenceencoding an ERR alpha polypeptide or fragment thereof. An exemplaryhuman ERR alpha nucleic acid sequence is provided at NCBI Ref:NM_001282450 and reproduced below:

(SEQ ID NO: 13) 1 tagaggtctc ccgcgggcgg ggagggggag gcgtagcaactttaggcaac ttcccaaagg 61 tgtgcgcagg ttgggggcgg gacgcggcgc cccgggaggtggcggcctct gcgacagcgg 121 gagtataaga gtggacctgc aggctggtcg cgaggaggtggagcggcgcc cgccgtgtgc 181 ctgggaccgg catgctgggg caggagggca gccgcgtgtcaggtgaccag cgccatgtcc 241 agccaggtgg tgggcattga gcctctctac atcaaggcagagccggccag ccctgacagt 301 ccaaagggtt cctcggagac agagaccgag cctcctgtggccctggcccc tggtccagct 361 cccactcgct gcctcccagg ccacaaggaa gaggaggatggggagggggc tgggcctggc 421 gagcagggcg gtgggaagct ggtgctcagc tccctgcccaagcgcctctg cctggtctgt 481 ggggacgtgg cctccggcta ccactatggt gtggcatcctgtgaggcctg caaagccttc 541 ttcaagagga ccatccaggg gagcatcgag tacagctgtccggcctccaa cgagtgtgag 601 atcaccaagc ggagacgcaa ggcctgccag gcctgccgcttcaccaagtg cctgcgggtg 661 ggcatgctca aggagggagt gcgcctggac cgcgtccggggtgggcggca gaagtacaag 721 cggcggccgg aggtggaccc actgcccttc ccgggccccttccctgctgg gcccctggca 781 gtcgctggag gcccccggaa gacagcagcc ccagtgaatgcactggtgtc tcatctgctg 841 gtggttgagc ctgagaagct ctatgccatg cctgaccccgcaggccctga tgggcacctc 901 ccagccgtgg ctaccctctg tgacctcttt gaccgagagattgtggtcac catcagctgg 961 gccaagagca tcccaggctt ctcatcgctg tcgctgtctgaccagatgtc agtactgcag 1021 agcgtgtgga tggaggtgct ggtgctgggt gtggcccagcgctcactgcc actgcaggat 1081 gagctggcct tcgctgagga cttagtcctg gatgaagagggggcacgggc agctggcctg 1141 ggggaactgg gggctgccct gctgcaacta gtgcggcggctgcaggccct gcggctggag 1201 cgagaggagt atgttctact aaaggccttg gcccttgccaattcagactc tgtgcacatc 1261 gaagatgccg aggctgtgga gcagctgcga gaagctctgcacgaggccct gctggagtat 1321 gaagccggcc gggctggccc cggagggggt gctgagcggcggcgggcggg caggctgctg 1381 ctcacgctac cgctcctccg ccagacagcg ggcaaagtgctggcccattt ctatggggtg 1441 aagctggagg gcaaggtgcc catgcacaag ctgttcttggagatgctcga ggccatgatg 1501 gactgaggca aggggtggga ctggtggggg ttctggcaggacctgcctag catggggtca 1561 gccccaaggg ctggggcgga gctggggtct gggcagtgccacagcctgct ggcagggcca 1621 gggcaatgcc atcagcccct gggaacaggc cccacgccctctcctccccc tcctaggggg 1681 tgtcagaagc tgggaacgtg tgtccaggct ctgggcacagtgctgcccct tgcaagccat 1741 aacgtgcccc cagagtgtag ggggccttgc ggaagccatagggggctgca cgggatgcgt 1801 gggaggcaga aacctatctc agggagggaa ggggatggaggccagagtct cccagtgggt 1861 gatgcttttg ctgctgctta atcctacccc ctcttcaaagcagagtggga cttggagagc 1921 aaaggcccat gcccccttcg ctcctcctct catcatttgcattgggcatt agtgtccccc 1981 cttgaagcaa taactccaag cagactccag cccctggacccctggggtgg ccagggcttc 2041 cccatcagct cccaacgagc ctcctcaggg ggtaggagagcactgcctct atgccctgca 2101 gagcaataac actatattta tttttgggtt tggccagggaggcgcaggga catggggcaa 2161 gccagggccc agagcccttg gctgtacaga gactctattttaatgtatat ttgctgcaaa 2221 gagaaaccgc ttttggtttt aaacctttaa tgagaaaaaaatatataata ccgagctcaa 2281 aaaaaaaaaa aaa

An exemplary murine ERR alpha nucleic acid sequence is provided at NCBIRef No. NM_007953.2:

(SEQ ID NO: 14) 1 tggaggaagc ggagtaggaa gcagccgcga tgtccttttgtgtcctacaa gcagccagcg 61 gcgccgccga gtgagggggg acgcagcgcg gcggggcggtgcggccggag gaggcggccc 121 ccgctcaccc cggcgctccg ggccgctcgg cccccatgcctgcccgccag ccctgccgga 181 gcccaaggtg accagcacca tgtccagcca ggtggtgggcatcgagcctc tctacatcaa 241 ggcagagcca gccagtcctg acagtccaaa gggttcctcagagactgaga ctgaaccccc 301 ggtgaccctg gcctctggtc cagctccagc ccgctgccttccagggcaca aggaggagga 361 ggatggggag ggggcagggt ctggtgagca gggcagtgggaagctagtgc tcagctctct 421 acccaaacgc ctctgcctgg tctgtgggga tgtggcctctggctaccact acggtgtggc 481 atcctgtgag gcctgcaaag ccttcttcaa gaggaccatccaggggagca tcgagtacag 541 ctgtccggcc tccaatgagt gtgagatcac caagcggagacgcaaggcct gtcaggcctg 601 ccgcttcacc aagtgcctgc gggtgggcat gctcaaggagggtgtgcgtc tggaccgtgt 661 ccgcggcgga cggcagaagt acaaacggcg gccagaggtggaccctttgc ctttcccggg 721 ccccttccct gctggacctc tggcagtagc tggaggacccaggaagacag ccccagtgaa 781 cgctctggtg tcgcatctgc tggtggttga acctgagaagctgtacgcca tgcctgaccc 841 agcaagcccc gatggacacc tccccgctgt ggccactctctgtgaccttt ttgatcgaga 901 gatagtggtc accatcagct gggccaagag catcccaggcttctcctcac tgtcactgtc 961 tgaccagatg tcagtactgc agagtgtgtg gatggaagtgctggtgctgg gtgtggccca 1021 gcgctcactg ccactgcagg atgagctggc ctttgctgaggacctggtcc tagatgaaga 1081 gggggcacgg gcagctggcc tgggggatct gggggcagccctgctgcagc tggttcggcg 1141 actgcaagct cttcggctgg agcgggagga gtacgtcctgctgaaagctc tggcccttgc 1201 caattctgac tctgtgcaca ttgaagatgc tgaggctgtggagcagctgc gcgaagccct 1261 gcatgaggcc ctgctggagt atgaagctgg ccgggctggccctggagggg gtgctgagcg 1321 gaggcgtgca ggcaggctgc tgcttacgct gccactcctccgccagacag caggcaaagt 1381 cctggcccat ttctatgggg tgaagctgga gggcaaggtgcccatgcaca agctgttttt 1441 ggaaatgctt gaggccatga tggactgagg caaggggtgggacagggtgg ggtggctggc 1501 aggatctgcc cagcataggg tgttagcccc aaaggggcaaagctggagtc tgggcagtgc 1561 catagcctgc tggcagggcc agggcaatgc catccgcccctgggagaagg cttcatgccc 1621 ttccctcccc actttgtgtg tgtgggggat tgtcagaagccaggaaagtg aatgcccagg 1681 tgtgggcaca gtgctgcccc ttgcaagcca taacgtgccccccaagagtg ttgggggcct 1741 cgcggaagcc atagggggct gcaggggatg tgcaggaggcagacactgat ctcagggagg 1801 gaagggatgg aggccgccgg ctcccactgg gtgatgcttttgctgctgct taatccgatc 1861 tcctctccgg agcagagggg ggcttggaaa gcaaaggccccgtcccttcg ctcctctcct 1921 catccgcatt gggcattatt gccccccctt gaagcaataactccaagcag gctccagccc 1981 ctggacccca ggggtggcca gggcccccta tcagctcccacctcaagggg tgggggacag 2041 cactgcctct atgccctgca gagcaataac actatatttatttttgggtt tggccaggga 2101 ggcgcagggc catggggcaa gccagggccc agagcccttggctgtacaga gactctattt 2161 taatgtatat ttgctgcaaa gagaaaccgc ttttggttttgaacctttaa tgagaaaaaa 2221 aatatactat ggagctcaag taaaaaaaaa aaaaaaaaaaaaaa

By “estrogen-related receptor (ERR) gamma polypeptide” also termed“ERRS” is meant a protein having at least 85% amino acid sequenceidentity to an estrogen-related receptor gamma sequence provided at NCBIRef No. P62508 (human), NP_001230721.1 (murine), or a fragment thereofhaving transcriptional regulatory activity.

The sequence of human ERR gamma is provided below:

sp|P62508|ERR3_HUMAN Estrogen-related receptor gamma OS═Homo sapiens GN

(SEQ ID NO: 15) MDSVELCLPE SFSLHYEEEL LCRMSNKDRH IDSSCSSFIKTEPSSPASLT DSVNHHSPGG SSDASGSYSS TMNGHQNGLDSPPLYPSAPI LGGSGPVRKL YDDCSSTIVE DPQTKCEYMLNSMPKRLCLV CGDIASGYHY GVASCEACKA FFKRTIQGNIEYSCPATNEC EITKRRRKSC QACRFMKCLK VGMLKEGVRLDRVRGGRQKY KRRIDAENSP YLNPQLVQPA KKPYNKIVSHLLVAEPEKIY AMPDPTVPDS DIKALTTLCD LADRELVVIIGWAKHIPGFS TLSLADQMSL LQSAWMEILI LGVVYRSLSFEDELVYADDY IMDEDQSKLA GLLDLNNAIL QLVKKYKSMKLEKEEFVTLK AIALANSDSM HIEDVEAVQK LQDVLHEALQDYEAGQHMED PRRAGKMLMT LPLLRQTSTK AVQHFYNIKL EGKVPMHKLF LEMLEAKVA murine estrogen-related receptor gamma sequence is provided at NCBIRef No. NP_001230721.1. The sequence of murine ERR gamma is providedbelow:

(SEQ ID NO: 16) MSNKDRHIDSSCSSFIKTEPSSPASLTDSVNHHSPGGSSDASGSYSSTMNGHQNGLDSPPLYPSAPILGGSGPVRKLYDDCSSTIVEDPQTKCEYMLNSMPKRLCLVCGDIASGYHYGVASCEACKAFFKRTIQGNIEYSCPATNECEITKRRRKSCQACRFMKCLKVGMLKEGVRLDRVRGGRQKYKRRIDAENSPYLNPQLLQSAWMEILILGVVYRSLSFEDELVYADDYIMDEDQSKLAGLLDLNNAILQLVKKYKSMKLEKEEFVTLKAIALANSDSMHIEDVEAVQKLQDVLHEALQDYEAGQHMEDPRRAGKMLMTLPLLRQTSTKAVQHFYNIKLEGKVPMH KLFLEMLEAKV

By “ERR gamma polynucleotide” is meant any nucleic acid sequenceencoding an ERR gamma polypeptide or fragment thereof. An exemplaryhuman ERR gamma nucleic acid sequence is provided at NCBI Ref:NM_001438.3

(SEQ ID NO: 17)aagctccaat cggggcttta agtccttgat taggagagtg tgagagcttt ggtcccaact 61ggctgtgcct ataggcttgt cactaggaga acatttgtgt taattgcact gtgctctgtc 121aaggaaactt tgatttatag ctggggtgca caaataatgg ttgccggtcg cacatggatt 181cggtagaact ttgccttcct gaatcttttt ccctgcacta cgaggaagag cttctctgca 241gaatgtcaaa caaagatcga cacattgatt ccagctgttc gtccttcatc aagacggaac 301cttccagccc agcctccctg acggacagcg tcaaccacca cagccctggt ggctcttcag 361acgccagtgg gagctacagt tcaaccatga atggccatca gaacggactt gactcgccac 421ctctctaccc ttctgctcct atcctgggag gtagtgggcc tgtcaggaaa ctgtatgatg 481actgctccag caccattgtt gaagatcccc agaccaagtg tgaatacatg ctcaactcga 541tgcccaagag actgtgttta gtgtgtggtg acatcgcttc tgggtaccac tatggggtag 601catcatgtga agcctgcaag gcattcttca agaggacaat tcaaggcaat atagaataca 661gctgccctgc cacgaatgaa tgtgaaatca caaagcgcag acgtaaatcc tgccaggctt 721gccgcttcat gaagtgttta aaagtgggca tgctgaaaga aggggtgcgt cttgacagag 781tacgtggagg tcggcagaag tacaagcgca ggatagatgc ggagaacagc ccatacctga 841accctcagct ggttcagcca gccaaaaagc catataacaa gattgtctca catttgttgg 901tggctgaacc ggagaagatc tatgccatgc ctgaccctac tgtccccgac agtgacatca 961aagccctcac tacactgtgt gacttggccg accgagagtt ggtggttatc attggatggg 1021cgaagcatat tccaggcttc tccacgctgt ccctggcgga ccagatgagc cttctgcaga 1081gtgcttggat ggaaattttg atccttggtg tcgtataccg gtctctttcg tttgaggatg 1141aacttgtcta tgcagacgat tatataatgg acgaagacca gtccaaatta gcaggccttc 1201ttgatctaaa taatgctatc ctgcagctgg taaagaaata caagagcatg aagctggaaa 1261aagaagaatt tgtcaccctc aaagctatag ctcttgctaa ttcagactcc atgcacatag 1321aagatgttga agccgttcag aagcttcagg atgtcttaca tgaagcgctg caggattatg 1381aagctggcca gcacatggaa gaccctcgtc gagctggcaa gatgctgatg acactgccac 1441tcctgaggca gacctctacc aaggccgtgc agcatttcta caacatcaaa ctagaaggca 1501aagtcccaat gcacaaactt tttttggaaa tgttggaggc caaggtctga ctaaaagctc 1561cctgggcctt cccatccttc atgttgaaaa agggaaaata aacccaagag tgatgtcgaa 1621gaaacttaga gtttagttaa caacatcaaa aatcaacaga ctgcactgat aatttagcag 1681caagactatg aagcagcttt cagattcctc cataggttcc tgatgagttt ctttctactt 1741tctccatcat cttctttcct ctttcttccc acatttctct ttctctttat tttttctcct 1801tttcttcttt cacctccctt atttctttgc ttctttcatt cctagttccc attctccttt 1861attttcttcc cgtctgcctg ccttctttct tttctttacc tactctcatt cctctctttt 1921ctcatccttc cccttttttc taaatttgaa atagctttag tttaaaaaaa aatcctccct 1981tccccctttc ctttcccttt ctttcctttt tccctttcct tttccctttc ctttcctttc 2041ctcttgacct tctttccatc tttctttttc ttccttctgc tgctgaactt ttaaaagagg 2101tctctaactg aagagagatg gaagccagcc ctgccaaagg atggagatcc ataatatgga 2161tgccagtgaa cttattgtga accatactgt ccccaatgac taaggaatca aagagagaga 2221accaacgttc ctaaaagtac agtgcaacat atacaaattg actgagtgca gtattagatt 2281tcatgggagc agcctctaat tagacaactt aagcaacgtt gcatcggctg cttcttatca 2341ttgcttttcc atctagatca gttacagcca tttgattcct taattgtttt ttcaagtctt 2401ccaggtattt gttagtttag ctactatgta actttttcag ggaatagttt aagctttatt 2461cattcatgca atactaaaga gaaataagaa tactgcaatt ttgtgctggc tttgaacaat 2521tacgaacaat aatgaaggac aaatgaatcc tgaaggaaga tttttaaaaa tgttttgttt 2581cttcttacaa atggagattt ttttgtacca gctttaccac ttttcagcca tttattaata 2641tgggaattta acttactcaa gcaatagttg aagggaaggt gcatattatc acggatgcaa 2701tttatgttgt gtgccagtct ggtcccaaac atcaatttct taacatgagc tccagtttac 2761ctaaatgttc actgacacaa aggatgagat tacacctaca gtgactctga gtagtcacat 2821atataagcac tgcacatgag atatagatcc gtagaattgt caggagtgca cctctctact 2881tgggaggtac aattgccata tgatttctag ctgccatggt ggttaggaat gtgatactgc 2941ctgtttgcaa agtcacagac cttgcctcag aaggagctgt gagccagtat tcatttaaga 3001ggcaataagg caaatgccag aattaaaaaa aaaaatcatc aaagacagaa aatgcctgac 3061caaattctaa aacctaatcc atataagttt attcatttag gaatgttcgt ttaaattaat 3121ctgcagtttt taccaagagc taagccaata tatgtgcttt tcaaccagta ttgtcacagc 3181atgaaagtca agtcaggttc cagactgtta agaggtgtaa tctaatgaag aaatcaatta 3241gatgccccga aatctacagt cgctgaataa ccaataaaca gtaacctcca tcaaatgcta 3301taccaatgga ccagtgttag tagctgctcc ctgtattatg tgaacagtct tattctatgt 3361acacagatgt aattaaaatt gtaatcctaa caaacaaaag aaatgtagtt cagcttttca 3421atgtttcatg tttgctgtgc ttttctgaat tttatgttgc attcaaagac tgttgtcttg 3481ttcttgtggt gtttggattc ttgtggtgtg tgcttttaga cacagggtag aattagagac 3541aatattggat gtacaattcc tcaggagact acagtagtat attctattcc ttaccagtaa 3601taaggttctt cctaataata attaagagat tgaaactcca aacaagtatt cattatgaac 3661agatacacat caaaatcata ataatatttt caaaacaagg aataatttct ctaatggttt 3721attatagaat accaatgtat agcttagaaa taaaactttg aatatttcaa gaatatagat 3781aagtctaatt tttaaatgct gtatatatgg ctttcactca atcatctctc agatgttgtt 3841attaactcgc tctgtgttgt tgcaaaactt tttggtgcag attcgtttcc aaaactattg 3901ctactttgtg tgctttaaac aaaatacctt gggttgatga aacatcaacc cagtgctagg 3961aatactgtgt atctatcatt agctatatgg gactatattg tagattgtgg tttctcagta 4021gagaagtgac tgtagtgtga ttctagataa atcatcatta gcaattcatt cagatggtca 4081ataacttgaa atttatagct gtgataggag ttcagaaatt ggcacatccc tttaaaaata 4141acaacagaaa atacaactcc tgggaaaaaa ggtgctgatt ctataagatt atttatatat 4201gtaagtgttt aaaaagatta ttttccagaa agtttgtgca gggtttaagt tgctactatt 4261caactacact atatataaat aaaatatata caatatatac attgttttca ctgtatcaca 4321ttaaagtact tgggcttcag aagtaagagc caaccaactg aaaacctgag atggagatat 4381gttcaaagaa tgagatacaa ttttttagtt ttcagtttaa gtaactctca gcattacaaa 4441agagtaagta tctcacaaat aggaaataaa actaaaacgt ggatttaaaa agaactgcac 4501gggctttagg gtaaatgctc atcttaaacc tcactagagg gaagtcttct caagtttcaa 4561gcaagaccat ttacttaatg tgaagttttg gaaagttata aaggtgtatg ttttagccat 4621atgattttaa ttttaatttt gcttctttta ggttcgttct tatttaaagc aatatgattg 4681tgtgactcct tgtagttaca cttgtgtttc aatcagatca gattgttgta tttattccac 4741tattttgcat ttaaatgata acataaaaga tataaaaaat ttaaaactgc tatttttctt 4801atagaagaga aaatgggtgt tggtgattgt attttaatta tttaagcgtc tctgtttacc 4861tgcctaggaa aacattttat ggcagtctta tgtgcaaaga tcgtaaaagg acaaaaaatt 4921taaactgctt ataataatcc aggagttgca ttatagccag tagtaaaaat aataataata 4981ataataaaac catgtctata gctgtagatg ggcttcacat ctgtaaagca atcaattgta 5041tatttttgtg atgtgtacca tactgtgtgc tccagcaaat gtccatttgt gtaaatgtat 5101ttattttata ttgtatatat tgttaaatgc aaaaaggaga tatgattctg taactccaat 5161cagttcagat gtgtaactca aattattatg cctttcagga tgatggtaga gcaatattaa 5221acaagcttcc

By “ERR gamma polynucleotide” is meant any nucleic acid sequenceencoding an ERR gamma polypeptide or fragment thereof. An exemplarymurine ERR gamma nucleic acid sequence is provided at NCBI Ref:NM_001243792.1 and reproduced below:

(SEQ ID NO: 18) 1agcccgaacc ccgtgcccga ttcctggtgc ggagtgcgag aggttcccgc ggcgcctggc 61ggacagtctc gctggcctcc ggtgacttgt tttgtgttgg ttttcccctc ttgcagccgg 121cgaccaagcg gacatcctcg gggaccccca aagccaccca ctcccgagag ctcggagagc 181ggctctgcac gagggacctt agctacttgc tggttcatca atgaagcaac ccgaagtgat 241gaagatgtaa ggaacgcatc ctacgctagc actgttgcag ttggaaaggc ttctctgcag 301aatgtcaaac aaagatcgac acattgattc cagctgttcg tccttcatca agacggaacc 361ctccagccca gcctccctga cggacagcgt caaccaccac agccctggtg ggtcttccga 421cgccagtggg agttacagtt caaccatgaa tggccatcag aacggactgg actcgccacc 481tctctacccc tctgctccga tcctgggagg cagcgggcct gtccggaaac tgtatgatga 541ctgctccagc accatcgtag aggatcccca gaccaagtgt gaatatatgc tcaactccat 601gcccaagaga ctgtgcttag tgtgtggcga catcgcctct gggtaccact atggggttgc 661atcatgtgaa gcctgcaagg cattcttcaa gaggacgatt caaggtaaca tagagtacag 721ctgcccagcc acgaatgaat gtgagatcac aaagcgcaga cgcaaatcct gccaggcctg 781ccgcttcatg aagtgtctca aagtgggcat gctgaaagaa ggggtccgtc ttgacagagt 841gcgtggaggt cggcagaagt acaagcgcag aatagatgct gagaacagcc catacctgaa 901ccctcagctg gtgcagccag ccaaaaagcc atataacaag attgtctcgc atttgttggt 961ggctgaacca gagaagatct atgccatgcc tgaccctact gtccccgaca gtgacatcaa 1021agccctcacc acactctgtg acttggctga ccgagagttg gtggttatca ttggatgggc 1081aaaacatatt ccaggcttct ccacactgtc cctggcagac cagatgagcc tcctccagag 1141tgcatggatg gagattctga tcctcggcgt tgtgtaccga tcgctttcgt ttgaggatga 1201acttgtctat gcagacgatt atataatgga tgaagaccag tctaaattag caggccttct 1261tgacctaaat aatgctatcc tgcagctggt gaagaagtac aagagcatga agctagagaa 1321ggaagaattc gtcaccctca aagcaatagc tcttgctaat tcagattcca tgcatataga 1381agatgtggaa gctgtgcaga aacttcagga tgtgttacat gaggccctgc aggattacga 1441ggctggccag cacatggaag accctcgccg tgcaggcaag atgctgatga cgctgccgct 1501gctgaggcag acctccacca aggcagtcca gcacttctac aacatcaaac tcgaaggcaa 1561agtgcccatg cacaaacttt ttttggaaat gctggaggcc aaggtctgac taaaagcccc 1621ccctgggccc tcccatcctg cacgttgaaa agggaagata aacccaagaa tgatgtcgaa 1681gaatcttaga gtttagtgaa caacattaaa aatcaacaga ctgcactgat attttagcag 1741ccacagtacg atgcagcctg cggattccgc tacatcttcc tgataggttt cctctacttt 1801atcccacgat cctctggcca catccctgca ttcctccact cttccttgtt ctattattat 1861gtttggcttc tttcactaat agttcatttt ccctcctccc ctcccttctc ttctccctcc 1921ctcctctgtc tcccccttcc ttcctttctc ttcctttcca caatcttctc ctcttgcctt 1981gctctcacct ctcttcgctt tctcacatct cctcccactc tgcgtacata gtcaatacct 2041ctgattgtat ggaacatttc ttttacctct tgcatctctt ctccgtctct tccttcccca 2101cttttttttg tttgtttgtt tgtttccttt ccttccttct gctgctgaac tcttaatagc 2161agtctctaac tggagagaga aagagagaga gatggaagcc agccctgcca aaggacagag 2221atccatacta tggatgccag tgaacttgtc atgaaccatg acatccccag tgagtaagga 2281atcaaagaga gaaccgtacc taaagtacat tgcaacgcaa acggatcaac ttagtgcagt 2341attagattct accgggcagc cttcgatcag acaacctaag tggcggcatt ggctgcttct 2401ccttgctttc tcatctagat cagttacagc catttgattc cttaattctt ttgtcaagtc 2461ttccaggtgt tggttagttt agctactatg taactttttc agggaatcct ttaagcttta 2521ttcattcatg caatactaga gaggggtaag gataccgcaa cctcgtgctg gctttgaaca 2581attgaacact aatgaaggac aaatgaaccc tgaaggaaga tttttaaaaa tgtttcgttt 2641cttcttacaa atggagattt ttttgtacca gctttaccac ttttcagcca tttattaata 2701tggggattta acttactcaa gcaatagttg aagggaaggt gcatattacc acggatgcaa 2761tttatgttgt gtgccagtct ggtcccaaac atcagtttct tacatgagct ccagtttgcc 2821taaatgttca ctgacaccaa ggattagatg atacctgccg tgacaccgag tggtcccatc 2881cacgagcact gcacatggga tccctatctg tagaattagc accagtacac ctccctgccg 2941ggagggacag tcgccatacg gtttctagct gccctcgtgg ttaggaacaa gatgctgcct 3001gtatacaaac tctgtctcag aaggagctgt gagccaatac catttcagag gcaataaagg 3061ctaagtgcca gaattcaaac caaccaacca tcaaagacag cagacgcctg accaaattct 3121aaagtcctga tccataggag tcgattcact taggaatggt tgtttaaatt aacctgcagg 3181tttgttttgt ttccttgttt gtttttttac caaaagctaa gccaatagat gtgctttttc 3241aacaagtatg gtcacagcac gaaggtcagt caggtttcag actgtaacca ggtgtaatct 3301aatgaagaaa tcaaatgtcc cctcccgaaa cctacagtcg ccgaataacc agaaaccagt 3361aacctccgta gaacgcttta ccaatggacc agtgttagta gctgctctct gtattctgtg 3421gacagtctta ttctatgtac acagatgtaa ttaaagttgt actcctaaca aacaaaagaa 3481tagttcagct tcaatgttcc atgtttgctg cgcttttctg aactttatgt tgcattcaga 3541aactgtcgtc ttgttctcgt ggtgtttgga ttcttgtggt gtgtgctttt agacacaggg 3601tagaattaga gacagtattg gatgtatact tcctcaggag actacagtag tatattctac 3661tccttaccag taataactaa gagattgaaa ctccaaaaca gtattcatta cgatcagaca 3721cacatcaaaa tcataataat attttcaaaa aagggataat ttctctaatg gtttattata 3781gaataccaat gtatagctta gacataaaac tttgaatatt caagaatata gataagtcta 3841atttttaaat gctgtatata aggcttccac ctgatcatct ctcagatgtt gttattaact 3901cgctctgtgt tgttgcaaac ctttttggtg cggacttgct tccaaaacta ttgctacttt 3961gtgtgcgtta agcaaaatac cttggactga gggtgtctca gccctgtgct aggaatactg 4021tgtatctatc attagctata tgggaatata tcgtagattg tggttctcag tagagaaagt 4081gactgtagtg tgactctagg taaatcatca ttagcaattc attcggatgg tcaataactt 4141gaaattgata gctgtgataa gttttaaaaa attggcaaat ccctgactaa acatcaacag 4201aaaatacaac tcctgggggg gaaaggtgct catcctgtaa gattctttca tcatgtaagt 4261gtttgaaaca ttactttgca gaaggtttat gcagggttta agttactacc gctcaataat 4321gctatatata cacaaatgga atatagacaa tgtatgtacc caccgtttca ctgagtcgca 4381gagaagaatc tgagcttcag aagccagagc ccacaagtga tcaggtgaga cagaggcaca 4441tttaaggaag gaggtacaat gtgtagttct ccgtttaaaa gacttggcct tttaaaacaa 4501caaatatctc acaactatgg tgaaaacaac aacagcttca agtgtggatc taaaggaaac 4561gcacaggttt agggtaaata ccatttgtac cttgctcgag caaagtttat tgttttgttt 4621ttttttgttt tgttttgttt tgttttcaag tttccagcaa gaccgtttag ttaatgccag 4681ctgtcaggaa gataccaagg tgtatgtttt agccatgcaa tttgcagttt tattttcctt 4741ttaggtttgt ccttatttaa ggcagtgcga ttgttttggc ttcttgtagt gactctcgtg 4801ttttaatcaa gccagattgt tgtatttatt ccactatttt gcatttaaat gatgacataa 4861aagatataaa aaatttaaaa ctgctatttt tcttatagaa gagaaaatgg atgttggtga 4921ttgtatttta attatttaag catctctgtt tacctgcctg ggacaacatt ttatggcagt 4981cttatgtgca aagatcgtga atggacaaaa caaaaaatta aactgcttac aatgatccag 5041gagttgcatt atagccagta gtaaaaataa taatgataat taataataat taataataat 5101aatgaaacca tgtctatagc tgtaggtggg catcacatct gtaaagcaat caattgtata 5161tttttgtgat gtgtaccata ctgtgtgctc cagcaaatgt ccatttgtgt aaatgtattt 5221attttatatt gtatatattg ttaaatgcaa aaaggagcta tgattctgtg actccaatca 5281gttcagatat gtaactcaaa ttattatgcc tttcaggagg atggtagaac aatattaaac 5341aagcttccac ttttaaaaaa aaaaaaaaaa aaaa

The invention provides for the use of other estrogen-related receptors,such as ERRbeta. The amino acid sequence of Homo sapiensestrogen-related receptor beta (ESRRbeta) is provided, for example, atNCBI Accession No. NP_004443, which is reproduced below:

(SEQ ID NO: 19) 1 mssddrhlgs scgsfiktep sspssgidal shhspsgssdasggfglalg thangldspp 61 mfagaglggt perksyedca sgimedsaik ceymlnaipkrlclvcgdia sgyhygvasc 121 eackaffkrt iqgnieyscp atneceitkr rrkscqacrfmkclkvgmlk egvrldrvrg 181 grqkykrrld sesspylslq isppakkplt kivsyllvaepdklyamppp gmpegdikal 241 ttlcdladre lvviigwakh ipgfsslslg dqmsllqsawmeililgivy rslpyddklv 301 yaedyimdee hsrlagllel yrailqlvrr ykklkvekeefvtlkalala nsdsmyiedl 361 eavqklqdll healqdyels qrheepwrtg kllltlpllrqtaakavqhf ysvklqgkvp 421 mhklflemle akvggeglrg spkdermssh dgkcpfqsaaftsrdqsnsp gipnprpssp 481 tpinergrqi spstrtpggq gkhlwltm

A polynucleotide sequence encoding an ERRbeta is provided, for example,at NCBI Accession No. NM_004452, which is reproduced below:

(SEQ ID NO: 20) 1 ccgcagagag gtgtggtcag ggacatttcc cctggccgggagcccatgga gcactgtcct 61 cagagatgcg caggttaggc tcactgtcta ggccaggcccaccttagtca ctgtggactg 121 gcaatggaag ctcttcctgg acacacctgc cctagccctcaccctggggt ggaagagaaa 181 tgagcttggc ttgcaactca gaccattcca cggaggcatcctccccttcc tgggctggtg 241 aataaaagtt tcctgaggtc aaggacttcc ttttccctgccaaaatggtg tccagaactt 301 tgaggccaga ggtgatccag tgatttggga gctgcaggtcacacaggctg ctcagagggc 361 tgctgaacag gatgtcctcg gacgacaggc acctgggctccagctgcggc tccttcatca 421 agactgagcc gtccagcccg tcctcgggca tcgatgccctcagccaccac agccccagtg 481 gctcgtccga cgccagcggc ggctttggcc tggccctgggcacccacgcc aacggtctgg 541 actcgccacc catgtttgca ggcgccgggc tgggaggcaccccatgccgc aagagctacg 601 aggactgtgc cagcggcatc atggaggact cggccatcaagtgcgagtac atgctcaacg 661 ccatccccaa gcgcctgtgc ctcgtgtgcg gggacattgcctctggctac cactacggcg 721 tggcctcctg cgaggcttgc aaggccttct tcaagaggactatccaaggg aacattgagt 781 acagctgccc ggccaccaac gagtgcgaga tcaccaaacggaggcgcaag tcctgccagg 841 cctgccgctt catgaaatgc ctcaaagtgg ggatgctgaaggaaggtgtg cgccttgatc 901 gagtgcgtgg aggccgtcag aaatacaagc gacggctggactcagagagc agcccatacc 961 tgagcttaca aatttctcca cctgctaaaa agccattgaccaagattgtc tcatacctac 1021 tggtggctga gccggacaag ctctatgcca tgcctccccctggtatgcct gagggggaca 1081 tcaaggccct gaccactctc tgtgacctgg cagaccgagagcttgtggtc atcattggct 1141 gggccaagca catcccaggc ttctcaagcc tctccctgggggaccagatg agcctgctgc 1201 agagtgcctg gatggaaatc ctcatcctgg gcatcgtgtaccgctcgctg ccctatgacg 1261 acaagctggt gtacgctgag gactacatca tggatgaggagcactcccgc ctcgcggggc 1321 tgctggagct ctaccgggcc atcctgcagc tggtacgcaggtacaagaag ctcaaggtgg 1381 agaaggagga gtttgtgacg ctcaaggccc tggccctcgccaactccgat tccatgtaca 1441 tcgaggatct agaggctgtc cagaagctgc aggacctgctgcacgaggca ctgcaggact 1501 acgagctgag ccagcgccat gaggagccct ggaggacgggcaagctgctg ctgacactgc 1561 cgctgctgcg gcagacggcc gccaaggccg tgcagcacttctatagcgtc aaactgcagg 1621 gcaaagtgcc catgcacaaa ctcttcctgg agatgctggaggccaaggtt ggccaagagc 1681 agcttagagg atctcccaag gatgaaagaa tgtcaagccatgatggaaaa tgccccttcc 1741 aatcagctgc cttcacaagc agggatcaga gcaactccccggggatcccc aatccacgcc 1801 cttctagtcc aacccccctc aatgagagag gcaggcagatctcacccagc actaggacac 1861 caggaggcca gggaaagcat ctctggctca ccatgtaacatctggcttgg agcaagtggg 1921 tgttctgcac accaggcagc tgcacctcac tggatctagtgttgctgcga gtgacctcac 1981 ttcagagccc ctctagcaga gtggggcgga agtcctgatggttggtgtcc atgaggtgga 2041 agctgctttt atacttaaaa ctcagatcac aacaggaaatgtgtcagtaa caatggaact 2101 ccatccaatg ggaaagttcc tggtactgaa ggggtccattggacactcag aaaagaagtt 2161 caggggccaa cttcttagct ggaatcctgg ccagatgaggaccctctccg gggaagggag 2221 aggactgact tagtggaagg tggtgaagtg aggagagtttaggggaacct tcccccagtg 2281 gaacagatct caagtttacc ctaaacctgc catttctggaaaatctgtaa agaggaaaca 2341 gcctgtctca gctgtactct catgatacag gtcatttgaaatgaaccaag aaataaaaca 2401 tgaaaatcca accatggaga aggtggtatg gctgggttttgtttggtccc cttgtcctta 2461 tacgttctaa agtttccaga ctggctttgt cactttgtgaactcgtcatg tgtgaaaacc 2521 aatctttgca tatagggaac ttcctcgggc cacactttaagaaccaagta agaggctctc 2581 aagactccag cagagtcggg aggccatggc agcgccttagaggagctgga acctgcaccc 2641 acctgtgtcg gtgggggggg cctcctttcc ccatagactctgccctccct ctgtgcagat 2701 ggaagtggca ggggagggtg accagcttgt gacaagaagactgaagggtc cagagtccat 2761 gctcacggaa cagcaccaaa gaaaagcact atgtggaaagattgttttat tttctaataa 2821 tgataatatg gctggaatgg cttcttaaga tgtatatattttttaaaatg gcagttcccc 2881 attgcagcat cacctacttg tatgtctttc tgcctctgtatatgttctcc cagaaacccc 2941 catgtaaatc aaatgcccta ggatgcttcc atcctggtcccatgtatctg gaatctaata 3001 aataaggaaa ggaaaaaaaa aaaaaaaaa

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

By “increases or decreases” is meant a positive or negative alteration.Such alterations are by 5%, 10%, 25%, 50%, 75%, 85%, 90% or even by 100%of a reference value. The terms “isolated,” “purified,” or “biologicallypure” refer to material that is free to varying degrees from componentswhich normally accompany it as found in its native state. “Isolate”denotes a degree of separation from original source or surroundings.“Purify” denotes a degree of separation that is higher than isolation. A“purified” or “biologically pure” protein is sufficiently free of othermaterials such that any impurities do not materially affect thebiological properties of the protein or cause other adverseconsequences. That is, a nucleic acid or peptide of this invention ispurified if it is substantially free of cellular material, viralmaterial, or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when chemically synthesized.Purity and homogeneity are typically determined using analyticalchemistry techniques, for example, polyacrylamide gel electrophoresis orhigh performance liquid chromatography. The term “purified” can denotethat a nucleic acid or protein gives rise to essentially one band in anelectrophoretic gel. For a protein that can be subjected tomodifications, for example, phosphorylation or glycosylation, differentmodifications may give rise to different isolated proteins, which can beseparately purified.

By “isolated cell” is meant a cell that is separated from the molecularand/or cellular components that naturally accompany the cell. Inparticular embodiments, the cell is a Sca1−CD34− cell isolated from apopulation expressing Sca1 and/or CD34. In other embodiments, the cellis isolated from a population expressing Oct4, Sox2, Klf4 and cMyc.

By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) thatis free of the genes which, in the naturally-occurring genome of theorganism from which the nucleic acid molecule of the invention isderived, flank the gene. The term therefore includes, for example, arecombinant DNA that is incorporated into a vector; into an autonomouslyreplicating plasmid or virus; or into the genomic DNA of a prokaryote oreukaryote; or that exists as a separate molecule (for example, a cDNA ora genomic or cDNA fragment produced by PCR or restriction endonucleasedigestion) independent of other sequences. In addition, the termincludes an RNA molecule that is transcribed from a DNA molecule, aswell as a recombinant DNA that is part of a hybrid gene encodingadditional polypeptide sequence.

By an “isolated polypeptide” is meant a polypeptide of the inventionthat has been separated from components that naturally accompany it.Typically, the polypeptide is isolated when it is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, a polypeptide of the invention. An isolated polypeptideof the invention may be obtained, for example, by extraction from anatural source, by expression of a recombinant nucleic acid encodingsuch a polypeptide; or by chemically synthesizing the protein. Puritycan be measured by any appropriate method, for example, columnchromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By “Klf4 polypeptide” is meant a protein or fragment thereof having atleast 85% homology to the sequence provided at NCBI Ref NP_004226.3(human) or NP_034767.2 (mouse). An exemplary human Klf4 amino acidsequence is provided below:

(SEQ ID NO: 21) MRQPPGESDMAVSDALLPSFSTFASGPAGREKTLRQAGAPNNRWREELSHMKRLPPVLPGRPYDLAAATVATDLESGGAGAACGGSNLAPLPRRETEEENDLLDLDFILSNSLTHPPESVAATVSSSASASSSSSPSSSGPASAPSTCSFTYPIRAGNDPGVAPGGTGGGLLYGRESAPPPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVLKASLSAPGSEYGSPSVISVSKGSPDGSHPVVVAPYNGGPPRTCPKIKQEAVSSCTHLGAGPPLSNGHRPAAHDFPLGRQLPSRTTPTLGLEEVLSSRDCHPALPLPPGFHPHPGPNYPSFLPDQMQPQVPPLHYQELMPPGSCMPEEPKPKRGRRSWPRKRTATHTCDYAGCGKTYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCDRAFSRSDHLALHMKRHF″An exemplary Klf4 murine amino acid sequence is provided below:

(SEQ ID NO: 22) MRQPPGESDMAVSDALLPSFSTFASGPAGREKTLRPAGAPTNRWREELSHMKRLPPLPGRPYDLAATVATDLESGGAGAACSSNNPALLARRETEEFNDLLDLDFILSNSLTHQESVAATVTTSASASSSSSPASSGPASAPSTCSFSYPIRAGGDPGVAASNTGGGLLYSRESAPPPTAPFNLADINDVSPSGGEVAELLRPELDPVYIPPQQPQPPGGGLMGKFVLKASLTTPGSEYSSPSVISVSKGSPDGSHPVVVAPYSGGPPRMCPKIKQEAVPSCTVSRSLEAHLSAGPQLSNGHRPNTHDFPLGRQLPTRTTPTLSPEELLNSRDCHPGLPLPPGFHPHPGPNYPPFLPDQMQSQVPSLHYQELMPPGSCLPEEPKPKRGRRSWPRKRTATHTCDYAGCGKTYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCDRAFSRSDHLALHMKRHF

By “Klf4” is meant a nucleic acid molecule encoding a Klf4 polypeptide.An exemplary human Klf4 polynucleotide sequence is provided atNM_004235.4 below:

(SEQ ID NO: 23) 1 agtttcccga ccagagagaa cgaacgtgtc tgcgggcgcgcggggagcag aggcggtggc 61 gggcggcggc ggcaccggga gccgccgagt gaccctcccccgcccctctg gccccccacc 121 ctcccacccg cccgtggccc gcgcccatgg ccgcgcgcgctccacacaac tcaccggagt 181 ccgcgccttg cgccgccgac cagttcgcag ctccgcgccacggcagccag tctcacctgg 241 cggcaccgcc cgcccaccgc cccggccaca gcccctgcgcccacggcagc actcgaggcg 301 accgcgacag tggtggggga cgctgctgag tggaagagagcgcagcccgg ccaccggacc 361 tacttactcg ccttgctgat tgtctatttt tgcgtttacaacttttctaa gaacttttgt 421 atacaaagga actttttaaa aaagacgctt ccaagttatatttaatccaa agaagaagga 481 tctcggccaa tttggggttt tgggttttgg cttcgtttcttctcttcgtt gactttgggg 541 ttcaggtgcc ccagctgctt cgggctgccg aggaccttctgggcccccac attaatgagg 601 cagccacctg gcgagtctga catggctgtc agcgacgcgctgctcccatc tttctccacg 661 ttcgcgtctg gcccggcggg aagggagaag acactgcgtcaagcaggtgc cccgaataac 721 cgctggcggg aggagctctc ccacatgaag cgacttcccccagtgcttcc cggccgcccc 781 tatgacctgg cggcggcgac cgtggccaca gacctggagagcggcggagc cggtgcggct 841 tgcggcggta gcaacctggc gcccctacct cggagagagaccgaggagtt caacgatctc 901 ctggacctgg actttattct ctccaattcg ctgacccatcctccggagtc agtggccgcc 961 accgtgtcct cgtcagcgtc agcctcctct tcgtcgtcgccgtcgagcag cggccctgcc 1021 agcgcgccct ccacctgcag cttcacctat ccgatccgggccgggaacga cccgggcgtg 1081 gcgccgggcg gcacgggcgg aggcctcctc tatggcagggagtccgctcc ccctccgacg 1141 gctcccttca acctggcgga catcaacgac gtgagcccctcgggcggctt cgtggccgag 1201 ctcctgcggc cagaattgga cccggtgtac attccgccgcagcagccgca gccgccaggt 1261 ggcgggctga tgggcaagtt cgtgctgaag gcgtcgctgagcgcccctgg cagcgagtac 1321 ggcagcccgt cggtcatcag cgtcagcaaa ggcagccctgacggcagcca cccggtggtg 1381 gtggcgccct acaacggcgg gccgccgcgc acgtgccccaagatcaagca ggaggcggtc 1441 tcttcgtgca cccacttggg cgctggaccc cctctcagcaatggccaccg gccggctgca 1501 cacgacttcc ccctggggcg gcagctcccc agcaggactaccccgaccct gggtcttgag 1561 gaagtgctga gcagcaggga ctgtcaccct gccctgccgcttcctcccgg cttccatccc 1621 cacccggggc ccaattaccc atccttcctg cccgatcagatgcagccgca agtcccgccg 1681 ctccattacc aagagctcat gccacccggt tcctgcatgccagaggagcc caagccaaag 1741 aggggaagac gatcgtggcc ccggaaaagg accgccacccacacttgtga ttacgcgggc 1801 tgcggcaaaa cctacacaaa gagttcccat ctcaaggcacacctgcgaac ccacacaggt 1861 gagaaacctt accactgtga ctgggacggc tgtggatggaaattcgcccg ctcagatgaa 1921 ctgaccaggc actaccgtaa acacacgggg caccgcccgttccagtgcca aaaatgcgac 1981 cgagcatttt ccaggtcgga ccacctcgcc ttacacatgaagaggcattt ttaaatccca 2041 gacagtggat atgacccaca ctgccagaag agaattcagtattttttact tttcacactg 2101 tcttcccgat gagggaagga gcccagccag aaagcactacaatcatggtc aagttcccaa 2161 ctgagtcatc ttgtgagtgg ataatcagga aaaatgaggaatccaaaaga caaaaatcaa 2221 agaacagatg gggtctgtga ctggatcttc tatcattccaattctaaatc cgacttgaat 2281 attcctggac ttacaaaatg ccaagggggt gactggaagttgtggatatc agggtataaa 2341 ttatatccgt gagttggggg agggaagacc agaattcccttgaattgtgt attgatgcaa 2401 tataagcata aaagatcacc ttgtattctc tttaccttctaaaagccatt attatgatgt 2461 tagaagaaga ggaagaaatt caggtacaga aaacatgtttaaatagccta aatgatggtg 2521 cttggtgagt cttggttcta aaggtaccaa acaaggaagccaaagttttc aaactgctgc 2581 atactttgac aaggaaaatc tatatttgtc ttccgatcaacatttatgac ctaagtcagg 2641 taatatacct ggtttacttc tttagcattt ttatgcagacagtctgttat gcactgtggt 2701 ttcagatgtg caataatttg tacaatggtt tattcccaagtatgccttaa gcagaacaaa 2761 tgtgtttttc tatatagttc cttgccttaa taaatatgtaatataaattt aagcaaacgt 2821 ctattttgta tatttgtaaa ctacaaagta aaatgaacattttgtggagt ttgtattttg 2881 catactcaag gtgagaatta agttttaaat aaacctataatattttatct gaaaaaaaaa 2941 aaaaaaaaa

An exemplary murine Klf4 polynucleotide sequence is provided atNM_010637.3 below:

(SEQ ID NO: 24) 1 agttccccgg ccaagagagc gagcgcggct ccgggcgcgcggggagcaga ggcggtggcg 61 ggcggcggcg gcacccggag ccgccgagtg cccctccccgcccctccagc cccccaccca 121 gcaacccgcc cgtgacccgc gcccatggcc gcgcgcacccggcacagtcc ccaggactcc 181 gcaccccgcg ccaccgccca gctcgcagtt ccgcgccaccgcggccattc tcacctggcg 241 gcgccgcccg cccaccgccc ggaccacagc ccccgcgccgccgacagcca cagtggccgc 301 gacaacggtg ggggacactg ctgagtccaa gagcgtgcagcctggccatc ggacctactt 361 atctgccttg ctgattgtct atttttataa gagtttacaacttttctaag aatttttgta 421 tacaaaggaa cttttttaaa gacatcgccg gtttatattgaatccaaaga agaaggatct 481 cgggcaatct gggggttttg gtttgaggtt ttgtttctaaagtttttaat cttcgttgac 541 tttggggctc aggtacccct ctctcttctt cggactccggaggaccttct gggcccccac 601 attaatgagg cagccacctg gcgagtctga catggctgtcagcgacgctc tgctcccgtc 661 cttctccacg ttcgcgtccg gcccggcggg aagggagaagacactgcgtc cagcaggtgc 721 cccgactaac cgttggcgtg aggaactctc tcacatgaagcgacttcccc cacttcccgg 781 ccgcccctac gacctggcgg cgacggtggc cacagacctggagagtggcg gagctggtgc 841 agcttgcagc agtaacaacc cggccctcct agcccggagggagaccgagg agttcaacga 901 cctcctggac ctagacttta tcctttccaa ctcgctaacccaccaggaat cggtggccgc 961 caccgtgacc acctcggcgt cagcttcatc ctcgtcttccccggcgagca gcggccctgc 1021 cagcgcgccc tccacctgca gcttcagcta tccgatccgggccgggggtg acccgggcgt 1081 ggctgccagc aacacaggtg gagggctcct ctacagccgagaatctgcgc cacctcccac 1141 ggcccccttc aacctggcgg acatcaatga cgtgagcccctcgggcggct tcgtggctga 1201 gctcctgcgg ccggagttgg acccagtata cattccgccacagcagcctc agccgccagg 1261 tggcgggctg atgggcaagt ttgtgctgaa ggcgtctctgaccacccctg gcagcgagta 1321 cagcagccct tcggtcatca gtgttagcaa aggaagcccagacggcagcc accccgtggt 1381 agtggcgccc tacagcggtg gcccgccgcg catgtgccccaagattaagc aagaggcggt 1441 cccgtcctgc acggtcagcc ggtccctaga ggcccatttgagcgctggac cccagctcag 1501 caacggccac cggcccaaca cacacgactt ccccctggggcggcagctcc ccaccaggac 1561 tacccctaca ctgagtcccg aggaactgct gaacagcagggactgtcacc ctggcctgcc 1621 tcttccccca ggattccatc cccatccggg gcccaactaccctcctttcc tgccagacca 1681 gatgcagtca caagtcccct ctctccatta tcaagagctcatgccaccgg gttcctgcct 1741 gccagaggag cccaagccaa agaggggaag aaggtcgtggccccggaaaa gaacagccac 1801 ccacacttgt gactatgcag gctgtggcaa aacctataccaagagttctc atctcaaggc 1861 acacctgcga actcacacag gcgagaaacc ttaccactgtgactgggacg gctgtgggtg 1921 gaaattcgcc cgctccgatg aactgaccag gcactaccgcaaacacacag ggcaccggcc 1981 ctttcagtgc cagaagtgtg acagggcctt ttccaggtcggaccaccttg ccttacacat 2041 gaagaggcac ttttaaatcc cacgtagtgg atgtgacccacactgccagg agagagagtt 2101 cagtattttt ttttctaacc tttcacactg tcttcccacgaggggaggag cccagctggc 2161 aagcgctaca atcatggtca agttcccagc aagtcagcttgtgaatggat aatcaggaga 2221 aaggaagagt tcaagagaca aaacagaaat actaaaaacaaacaaacaaa aaaacaaaca 2281 aaaaaaacaa gaaaaaaaaa tcacagaaca gatggggtctgatactggat ggatcttcta 2341 tcattccaat accaaatcca acttgaacat gcccggacttacaaaatgcc aaggggtgac 2401 tggaagtttg tggatatcag ggtatacact aaatcagtgagcttgggggg agggaagacc 2461 aggattccct tgaattgtgt ttcgatgatg caatacacacgtaaagatca ccttgtatgc 2521 tctttgcctt cttaaaaaaa aaaaaagcca ttattgtgtcggaggaagag gaagcgattc 2581 aggtacagaa catgttctaa cagcctaaat gatggtgcttggtgagtcgt ggttctaaag 2641 gtaccaaacg ggggagccaa agttctccaa ctgctgcatacttttgacaa ggaaaatcta 2701 gttttgtctt ccgatctaca ttgatgacct aagccaggtaaataagcctg gtttatttct 2761 gtaacatttt tatgcagaca gtctgttatg cactgtggtttcagatgtgc aataatttgt 2821 acaatggttt attcccaagt atgcctttaa gcagaacaaatgtgtttttc tatatagttc 2881 cttgccttaa taaatatgta atataaattt aagcaaacttctattttgta tatttgtaaa 2941 ctacaaagta aaaaaaaatg aacattttgt ggagtttgtattttgcatac tcaaggtgag 3001 aaataagttt taaataaacc tataatattt tatctgaacgacaaaaaaaa aaaaaaa

By “marker” is meant any protein or polynucleotide having an alterationin expression level or activity that is associated with a disease ordisorder.

By “negative” is meant that a cell expresses an undetectable level of amarker or a reduced level of marker, such that the cell can bedistinguished in a negative selection from a population of unselectedcells.

By “Oct4 polypeptide” is meant a protein or fragment thereof having atleast 85% homology to the sequence provided at NCBI Ref: NP_001167002.1(human) or NP_001239381.1 (murine) and having transcriptional regulatoryactivity.

An exemplary Oct4 human amino acid sequence is provided below:

(SEQ ID NO: 25) MGVLEGKVESQTTICRFEALQLSEKNMCKLRPLLQKWVEEADNNENLQEICKAETLVQARKRKRTSIENRVRGNLENLFLQCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQKGKRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPEGEAFPPVSVTTLGSPMHSN 

An exemplary Oct4 murine amino acid sequence (NCBI Ref: NP_001239381.1)is provided below:

(SEQ ID NO: 26) MKALQKELEQFAKLLKQKRITLGYTQADVGLTLGVLFGKVFSQTTICRFEALQLSLKNMCKLRPLLEKWVEEADNNENLQEICKSETLVQARKRKRTSIENRVRWSLETMFLKCPKPSLQQITHIANQLGLEKDVVRVWFCNRRQKGKRSSIEYSQREEYEATGTPFPGGAVSFPLPPGPHFGTPGYGSPHFTTLYSVPFPEGE AFPSVPVTALGSPMHSN

By “Oct4 polynucleotide” is meant a nucleic acid molecule encoding aOct4 polypeptide. An exemplary human Oct4 polynucleotide sequence isprovided at NM_001173531.2 and reproduced below:

(SEQ ID NO: 27) 1 ggaaaaaagg aaagtgcact tggaagagat ccaagtgggcaacttgaaga acaagtgcca 61 aatagcactt ctgtcatgct ggatgtcagg gctctttgtccactttgtat agccgctggc 121 ttatagaagg tgctcgataa atctcttgaa tttaaaaatcaattaggatg cctctatagt 181 gaaaaagata cagtaaagat gagggataat caatttaaaaaatgagtaag tacacacaaa 241 gcactttatc cattcttatg acacctgtta cttttttgctgtgtttgtgt gtatgcatgc 301 catgttatag tttgtgggac cctcaaagca agctggggagagtatatact gaatttagct 361 tctgagacat gatgctcttc ctttttaatt aacccagaacttagcagctt atctatttct 421 ctaatctcaa aacatcctta aactgggggt gatacttgagtgagagaatt ttgcaggtat 481 taaatgaact atcttctttt ttttttttct ttgagacagagtcttgctct gtcacccagg 541 ctggagtgca gtggcgtgat ctcagctcac tgcaacctccgcctcccggg ttcaagtgat 601 tctcctgcct cagcctcctg agtagctggg attacagtcccaggacatca aagctctgca 661 gaaagaactc gagcaatttg ccaagctcct gaagcagaagaggatcaccc tgggatatac 721 acaggccgat gtggggctca ccctgggggt tctatttgggaaggtattca gccaaacgac 781 catctgccgc tttgaggctc tgcagcttag cttcaagaacatgtgtaagc tgcggccctt 841 gctgcagaag tgggtggagg aagctgacaa caatgaaaatcttcaggaga tatgcaaagc 901 agaaaccctc gtgcaggccc gaaagagaaa gcgaaccagtatcgagaacc gagtgagagg 961 caacctggag aatttgttcc tgcagtgccc gaaacccacactgcagcaga tcagccacat 1021 cgcccagcag cttgggctcg agaaggatgt ggtccgagtgtggttctgta accggcgcca 1081 gaagggcaag cgatcaagca gcgactatgc acaacgagaggattttgagg ctgctgggtc 1141 tcctttctca gggggaccag tgtcctttcc tctggccccagggccccatt ttggtacccc 1201 aggctatggg agccctcact tcactgcact gtactcctcggtccctttcc ctgaggggga 1261 agcctttccc cctgtctccg tcaccactct gggctctcccatgcattcaa actgaggtgc 1321 ctgcccttct aggaatgggg gacaggggga ggggaggagctagggaaaga aaacctggag 1381 tttgtgccag ggtttttggg attaagttct tcattcactaaggaaggaat tgggaacaca 1441 aagggtgggg gcaggggagt ttggggcaac tggttggagggaaggtgaag ttcaatgatg 1501 ctcttgattt taatcccaca tcatgtatca cttttttcttaaataaagaa gcctgggaca 1561 cagtagatag acacacttaa aaaaaaaaa

An exemplary murine Oct4 polynucleotide sequence is provided atNM_001252452.1 and reproduced below:

(SEQ ID NO: 28) 1 gcagccttaa aacttcttca gaatagggtg acattttgtcctcagtgggg cggttttgag 61 taatctgtga gcagatagga acttgctggg tcccaggacatgaaagccct gcagaaggag 121 ctagaacagt ttgccaagct gctgaagcag aagaggatcaccttggggta cacccaggcc 181 gacgtggggc tcaccctggg cgttctcttt ggaaaggtgttcagccagac caccatctgt 241 cgcttcgagg ccttgcagct cagccttaag aacatgtgtaagctgcggcc cctgctggag 301 aagtgggtgg aggaagccga caacaatgag aaccttcaggagatatgcaa atcggagacc 361 ctggtgcagg cccggaagag aaagcgaact agcattgagaaccgtgtgag gtggagtctg 421 gagaccatgt ttctgaagtg cccgaagccc tccctacagcagatcactca catcgccaat 481 cagcttgggc tagagaagga tgtggttcga gtatggttctgtaaccggcg ccagaagggc 541 aaaagatcaa gtattgagta ttcccaacga gaagagtatgaggctacagg gacacctttc 601 ccaggggggg ctgtatcctt tcctctgccc ccaggtccccactttggcac cccaggctat 661 ggaagccccc acttcaccac actctactca gtcccttttcctgagggcga ggcctttccc 721 tctgttcccg tcactgctct gggctctccc atgcattcaaactgaggcac cagccctccc 781 tggggatgct gtgagccaag gcaagggagg tagacaagagaacctggagc tttggggtta 841 aattctttta ctgaggaggg attaaaagca caacaggggtggggggtggg atggggaaag 901 aagctcagtg atgctgttga tcaggagcct ggcctgtctgtcactcatca ttttgttctt 961 aaataaagac tgggacacac agtagatagc t

By “PGC1 alpha polypeptide” is meant a protein or fragment thereofhaving at least 85% identity to the amino acid sequence provided at NCBIRef: NP_037393.1 or UniProt Ref: Q9UBK2 (human), NCBI Ref: NP_032930.1(mouse) and having transcriptional coactivating activity. An exemplaryPGC1 alpha human amino acid sequence is provided below:

>sp|Q9UBK2|PRGC1_HUMAN Peroxisome proliferator-activated receptor gamma coactivator 1-alphaOS = Homo sapiens GN = PPARGC1A PE = 1 SV = 1 (SEQ ID NO: 29)MAWDMCNQDSESVWSDIECAALVGEDQPLCPDLPELDLSELDVNDLDTDSFLGGLKWCSDQSEIISNQYNNEPSNIFEKIDEENEANLLAVLTETLDSLPVDEDGLPSFDALTDGDVTTDNEASPSSMPDGTPPPQEAEEPSLLKKLLLAPANTQLSYNECSGLSTQNHANHNHRIRTNPAIVKTENSWSNKAKSICQQQKPQRRPCSELLKYLTTNDDPPHTKPTENRNSSRDKCTSKKKSHTQSQSQHLQAKPTTLSLPLTPESPNDPKGSPFENKTIERTLSVELSGTAGLTPPTTPPHKANQDNPFRASPKLKSSCKTVVPPPSKKPRYSESSGTQGNNSTKKGPEQSELYAQLSKSSVLTGGHEERKTKRPSLRLFGDHDYCQSINSKTEILINISQELQDSRQLENKDVSSDWQGQICSSTDSDQCYLRETLEASKQVSPCSTRKQLQDQEIRAELNKHFGHPSQAVFDDEADKTGELRDSDFSNEQFSKLPMFINSGLAMDGLFDDSEDESDKLSYPWDGTQSYSLFNVSPSCSSFNSPCRDSVSPPKSLFSQRPQRMRSRSRSFSRHRSCSRSPYSRSRSRSPGSRSSSRSCYYYESSHYRHRTHRNSPLYVRSRSRSPYSRRPRYDSYEEYQHERLKREEYRREYEKRESERAKQRERQRQKAIEERRVIYVGKIRPDTTRTELRDRFEVFGEIEECTVNLRDDGDSYGFITYRYTCDAFAALENGYTLRRSNETDFELYFCGRKQFFKSNYADLDSNSDDFDPASTKSKYDSLDFDSLLKEAQRSLRR 

An exemplary murine PGC1 alpha amino acid sequence is provided below:

(SEQ ID NO: 30) MAWDMCSQDSVWSDIECAALVGEDQPLCPDLPELDLSELDVNDLDTDSFLGGLKWCSDQSEIISNQYNNEPANIFEKIDEENEANLLAVLTETLDSLPVDEDGLPSFDALTDGAVTTDNEASPSSMPDGTPPPQEAEEPSLLKKLLLAPANTQLSYNECSGLSTQNHAANHTHRIRTNPAIVKTENSWSNKAKSICQQQKPQRRPCSELLKYLTTNDDPPHTKPTENRNSSRDKCASKKKSHTQPQSQHAQAKPTTLSLPLTPESPNDPKGSPFENKTIERTLSVELSGTAGLTPPTTPPHKANQDNPFKASPKLKPSCKTVVPPPTKRARYSECSGTQGSHSTKKGPEQSELYAQLSKSSGLSRGHEERKTKRPSLRLFGDHDYCQSLNSKTDILINISQELQDSRQLDFKDASCDWQGHICSSTDSGQCYLRETLEASKQVSPCSTRKQLQDQEIRAELNKHFGHPCQAVFDDKSDKTSELRDGDFSNEQFSKLPVFINSGLAMDGLFDDSEDESDKLSYPWDGTQPYSLFDVSPSCSSFNSPCRDSVSPPKSLFSQRPQRMRSRSRSFSRHRSCSRSPYSRSRSRSPGSRSSSRSCYYYESSHYRHRTHRNSPLYVRSRSRSPYSRRPRYDSYEAYEHERLKRDEYRKEHEKRESERAKQRERQKQKAIEERRVIYVGKIRPDTTRTELRDRFEVEGEIEECTVNLRDDGDSYGFITYRYTCDAFAALENGYTLRRSNETDFELYFCGRKQFFKSNYADLDTNSDDFDPASTKSKYDSLDFDSLLKEAQRSLRR

By “PGC1 alpha polynucleotide” is meant a nucleic acid molecule encodinga PGC1 alpha polypeptide. An exemplary human PGC1 alpha polynucleotidesequence is provided at NM_013261:

(SEQ ID NO: 31)tagtaagaca ggtgccttca gttcactctc agtaaggggc tggttgcctg catgagtgtg 61tgctctgtgt cactgtggat tggagttgaa aaagcttgac tggcgtcatt caggagctgg 121atggcgtggg acatgtgcaa ccaggactct gagtctgtat ggagtgacat cgagtgtgct 181gctctggttg gtgaagacca gcctctttgc ccagatcttc ctgaacttga tctttctgaa 241ctagatgtga acgacttgga tacagacagc tttctgggtg gactcaagtg gtgcagtgac 301caatcagaaa taatatccaa tcagtacaac aatgagcctt caaacatatt tgagaagata 361gatgaagaga atgaggcaaa cttgctagca gtcctcacag agacactaga cagtctccct 421gtggatgaag acggattgcc ctcatttgat gcgctgacag atggagacgt gaccactgac 481aatgaggcta gtccttcctc catgcctgac ggcacccctc caccccagga ggcagaagag 541ccgtctctac ttaagaagct cttactggca ccagccaaca ctcagctaag ttataatgaa 601tgcagtggtc tcagtaccca gaaccatgca aatcacaatc acaggatcag aacaaaccct 661gcaattgtta agactgagaa ttcatggagc aataaagcga agagtatttg tcaacagcaa 721aagccacaaa gacgtccctg ctcggagctt ctcaaatatc tgaccacaaa cgatgaccct 781cctcacacca aacccacaga gaacagaaac agcagcagag acaaatgcac ctccaaaaag 841aagtcccaca cacagtcgca gtcacaacac ttacaagcca aaccaacaac tttatctctt 901cctctgaccc cagagtcacc aaatgacccc aagggttccc catttgagaa caagactatt 961gaacgcacct taagtgtgga actctctgga actgcaggcc taactccacc caccactcct 1021cctcataaag ccaaccaaga taaccctttt agggcttctc caaagctgaa gtcctcttgc 1081aagactgtgg tgccaccacc atcaaagaag cccaggtaca gtgagtcttc tggtacacaa 1141ggcaataact ccaccaagaa agggccggag caatccgagt tgtatgcaca actcagcaag 1201tcctcagtcc tcactggtgg acacgaggaa aggaagacca agcggcccag tctgcggctg 1261tttggtgacc atgactattg ccagtcaatt aattccaaaa cagaaatact cattaatata 1321tcacaggagc tccaagactc tagacaacta gaaaataaag atgtctcctc tgattggcag 1381gggcagattt gttcttccac agattcagac cagtgctacc tgagagagac tttggaggca 1441agcaagcagg tctctccttg cagcacaaga aaacagctcc aagaccagga aatccgagcc 1501gagctgaaca agcacttcgg tcatcccagt caagctgttt ttgacgacga agcagacaag 1561accggtgaac tgagggacag tgatttcagt aatgaacaat tctccaaact acctatgttt 1621ataaattcag gactagccat ggatggcctg tttgatgaca gcgaagatga aagtgataaa 1681ctgagctacc cttgggatgg cacgcaatcc tattcattgt tcaatgtgtc tccttcttgt 1741tcttctttta actctccatg tagagattct gtgtcaccac ccaaatcctt attttctcaa 1801agaccccaaa ggatgcgctc tcgttcaagg tccttttctc gacacaggtc gtgttcccga 1861tcaccatatt ccaggtcaag atcaaggtct ccaggcagta gatcctcttc aagatcctgc 1921tattactatg agtcaagcca ctacagacac cgcacgcacc gaaattctcc cttgtatgtg 1981agatcacgtt caagatcgcc ctacagccgt cggcccaggt atgacagcta cgaggaatat 2041cagcacgaga ggctgaagag ggaagaatat cgcagagagt atgagaagcg agagtctgag 2101agggccaagc aaagggagag gcagaggcag aaggcaattg aagagcgccg tgtgatttat 2161gtcggtaaaa tcagacctga cacaacacgg acagaactga gggaccgttt tgaagttttt 2221ggtgaaattg aggagtgcac agtaaatctg cgggatgatg gagacagcta tggtttcatt 2281acctaccgtt atacctgtga tgcttttgct gctcttgaaa atggatacac tttgcgcagg 2341tcaaacgaaa ctgactttga gctgtacttt tgtggacgca agcaattttt caagtctaac 2401tatgcagacc tagattcaaa ctcagatgac tttgaccctg cttccaccaa gagcaagtat 2461gactctctgg attttgatag tttactgaaa gaagctcaga gaagcttgcg caggtaacat 2521gttccctagc tgaggatgac agagggatgg cgaatacctc atgggacagc gcgtccttcc 2581ctaaagacta ttgcaagtca tacttaggaa tttctcctac tttacactct ctgtacaaaa 2641acaaaacaaa acaacaacaa tacaacaaga acaacaacaa caataacaac aatggtttac 2701atgaacacag ctgctgaaga ggcaagagac agaatgatat ccagtaagca catgtttatt 2761catgggtgtc agctttgctt ttcctggagt ctcttggtga tggagtgtgc gtgtgtgcat 2821gtatgtgtgt gtgtatgtat gtgtgtggtg tgtgtgcttg gtttagggga agtatgtgtg 2881ggtacatgtg aggactgggg gcacctgacc agaatgcgca agggcaaacc atttcaaatg 2941gcagcagttc catgaagaca cgcttaaaac ctagaacttc aaaatgttcg tattctattc 3001aaaaggaaat atatatatat atatatatat atatatatat atatataaat taaaaaggaa 3061agaaaactaa caaccaacca accaaccaac caaccacaaa ccaccctaaa atgacagccg 3121ctgatgtctg ggcatcagcc tttgtactct gtttttttaa gaaagtgcag aatcaacttg 3181aagcaagctt tctctcataa cgtaatgatt atatgacaat cctgaagaaa ccacaggttc 3241catagaacta atatcctgtc tctctctctc tctctctctc tctctttttt ttttcttttt 3301ccttttgcca tggaatctgg gtgggagagg atactgcggg caccagaatg ctaaagtttc 3361ctaacatttt gaagtttctg tagttcatcc ttaatcctga cacccatgta aatgtccaaa 3421atgttgatct tccactgcaa atttcaaaag ccttgtcaat ggtcaagcgt gcagcttgtt 3481cagcggttct ttctgaggag cggacaccgg gttacattac taatgagagt tgggtagaac 3541tctctgagat gtgttcagat agtgtaattg ctacattctc tgatgtagtt aagtatttac 3601agatgttaaa tggagtattt ttattttatg tatatactat acaacaatgt tcttttttgt 3661tacagctatg cactgtaaat gcagccttct tttcaaaact gctaaatttt tcttaatcaa 3721gaatattcaa atgtaattat gaggtgaaac aattattgta cactaacata tttagaagct 3781gaacttactg cttatatata tttgattgta aaaacaaaaa gacagtgtgt gtgtctgttg 3841agtgcaacaa gagcaaaatg atgctttccg cacatccatc ccttaggtga gcttcaatct 3901aagcatcttg tcaagaaata tcctagtccc ctaaaggtat taaccacttc tgcgatattt 3961ttccacattt tcttgtcgct tgtttttctt tgaagtttta tacactggat ttgttagggg 4021aatuaaattt tctcatctaa aatttttcta uaauatatca tuattttatu taaautctct 4081caatgggtaa ccattaagaa atgtttttat tttctctatc aacagtagtt ttgaaactag 4141aagtcaaaaa tctttttaaa atgctgtttt gttttaattt ttgtgatttt aatttgatac 4201aaaatgctga ggtaataatt atagtatgat ttttacaata attaatgtgt gtctgaagac 4261tatctttgaa gccagtattt ctttcccttg gcagagtatg acgatggtat ttatctgtat 4321tttttacagt tatgcatcct gtataaatac tgatatttca ttcctttgtt tactaaagag 4381acatatttat cagttgcaga tagcctattt attataaatt atgagatgat gaaaataata 4441aagccagtgg aaattttcta cctaggatgc atgacaattg tcaggttgga gtgtaagtgc 4501ttcatttggg aaattcagct tttgcagaag cagtgtttct acttgcacta gcatggcctc 4561tgacgtgacc atggtgttgt tcttgatgac attgcttctg ctaaatttaa taaaaacttc 4621agaaaaacct ccattttgat catcaggatt tcatctgagt gtggagtccc tggaatggaa 4681ttcagtaaca tttggagtgt gtattcaagt ttctaaattg agattcgatt actgtttggc 4741tgacatgact tttctggaag acatgataca cctactactc aattgttctt ttcctttctc 4801tcgcccaaca cgatcttgta agatggattt cacccccagg ccaatgcagc taattttgat 4861agctgcattc atttatcacc agcatattgt gttctgagtg aatccactgt ttgtcctgtc 4921ggatgcttgc ttgatttttt ggcttcttat ttctaagtag atagaaagca ataaaaatac 4981tatgaaatga aagaacttgt tcacaggttc tgcgttacaa cagtaacaca tctttaatcc 5041gcctaattct tgttgttctg taggttaaat gcaggtattt taactgtgtg aacgccaaac 5101taaagtttac agtctttctt tctgaatttt gagtatcttc tgttgtagaa taataataaa 5161aagactatta agagcaataa attattttta agaaatcgag atttagtaaa tcctattatg 5221tgttcaagga ccacatgtgt tctctatttt gcctttaaat ttttgtgaac caattttaaa 5281tacattctcc tttttgccct ggattgttga catgagtgga atacttggtt tcttttctta 5341cttatcaaaa gacagcacta cagatatcat attgaggatt aatttatccc ccctaccccc 5401agcctgacaa atattgttac catgaagata gttttcctca atggacttca aattgcatct 5461agaattagtg gagcttttgt atcttctgca gacactgtgg gtagcccatc aaaatgtaag 5521ctgtgctcct ctcattttta tttttatttt tttgggagag aatatttcaa atgaacacgt 5581gcaccccatc atcactggag gcaaatttca gcatagatct gtaggatttt tagaagaccg 5641tgggccattg ccttcatgcc gtggtaagta ccacatctac aattttggta accgaactgg 5701tgctttagta atgtggattt ttttcttttt taaaagagat gtagcagaat aattcttcca 5761gtgcaacaaa atcaattttt tgctaaacga ctccgagaac aacagttggg ctgtcaacat 5821tcaaagcagc agagagggaa ctttgcacta ttggggtatg atgtttgggt cagttgataa 5881aaggaaacct tttcatgcct ttagatgtga gcttccagta ggtaatgatt atgtgtcctt 5941tcttgatggc tgtaatgaga acttcaatca ctgtagtcta agacctgatc tatagatgac 6001ctagaatagc catgtactat aatgtgatga ttctaaattt gtacctatgt gacagacatt 6061ttcaataatg tgaactgctg atttgatgga gctactttaa gatttgtagg tgaaagtgta 6121atactgttgg ttgaactatg ctgaagaggg aaagtgagcg attagttgag cccttgccgg 6181gccttttttc cacctgccaa ttctacatgt attgttgtgg ttttattcat tgtatgaaaa 6241ttcctgtgat tttttttaaa tgtgcagtac acatcagcct cactgagcta ataaagggaa 6301acgaatgttt caaatcta

An exemplary murine PGC1 alpha polynucleotide sequence is provided atNM_008904.2:

(SEQ ID NO: 32) 1 gtcatgtgac tggggactgt agtaagacag gtgccttcagttcactctca gtaaggggct 61 ggttgcctgc atgagtgtgt gctgtgtgtc agagtggattggagttgaaa aagcttgact 121 ggcgtcattc gggagctgga tggcttggga catgtgcagccaagactctg tatggagtga 181 catagagtgt gctgctctgg ttggtgagga ccagcctctttgcccagatc ttcctgaact 241 tgacctttct gaacttgatg tgaatgactt ggatacagacagctttctgg gtggattgaa 301 gtggtgtagc gaccaatcgg aaatcatatc caaccagtacaacaatgagc ctgcgaacat 361 atttgagaag atagatgaag agaatgaggc aaacttgctagcggttctca cagagacact 421 ggacagtctc cccgtggatg aagacggatt gccctcatttgatgcactga cagatggagc 481 cgtgaccact gacaacgagg ccagtccttc ctccatgcctgacggcaccc ctccccctca 541 ggaggcagaa gagccgtctc tacttaagaa gctcttactggcaccagcca acactcagct 601 cagctacaat gaatgcagcg gtcttagcac tcagaaccatgcagcaaacc acacccacag 661 gatcagaaca aaccctgcca ttgttaagac cgagaattcatggagcaata aagcgaagag 721 catttgtcaa cagcaaaagc cacaaagacg tccctgctcagagcttctca agtatctgac 781 cacaaacgat gaccctcctc acaccaaacc cacagaaaacaggaacagca gcagagacaa 841 atgtgcttcg aaaaagaagt cccatacaca accgcagtcgcaacatgctc aagccaaacc 901 aacaacttta tctcttcctc tgaccccaga gtcaccaaatgaccccaagg gttccccatt 961 tgagaacaag actattgagc gaaccttaag tgtggaactctctggaactg caggcctaac 1021 tcctcccaca actcctcctc ataaagccaa ccaagataaccctttcaagg cttcgccaaa 1081 gctgaagccc tcttgcaaga ccgtggtgcc accgccaaccaagagggccc ggtacagtga 1141 gtgttctggt acccaaggca gccactccac caagaaagggcccgagcaat ctgagttgta 1201 cgcacaactc agcaagtcct cagggctcag ccgaggacacgaggaaagga agactaaacg 1261 gcccagtctc cggctgtttg gtgaccatga ctactgtcagtcactcaatt ccaaaacgga 1321 tatactcatt aacatatcac aggagctcca agactctagacaactagact tcaaagatgc 1381 ctcctgtgac tggcaggggc acatctgttc ttccacagattcaggccagt gctacctgag 1441 agagactttg gaggccagca agcaggtctc tccttgcagcaccagaaaac agctccaaga 1501 ccaggaaatc cgagcggagc tgaacaagca cttcggtcatccctgtcaag ctgtgtttga 1561 cgacaaatca gacaagacca gtgaactaag ggatggcgacttcagtaatg aacaattctc 1621 caaactacct gtgtttataa attcaggact agccatggatggcctatttg atgacagtga 1681 agatgaaagt gataaactga gctacccttg ggatggcacgcagccctatt cattgttcga 1741 tgtgtcgcct tcttgctctt cctttaactc tccgtgtcgagactcagtgt caccaccgaa 1801 atccttattt tctcaaagac cccaaaggat gcgctctcgttcaagatcct tttctcgaca 1861 caggtcgtgt tcccgatcac catattccag gtcaagatcaaggtccccag gcagtagatc 1921 ctcttcaaga tcctgttact actatgaatc aagccactacagacaccgca cacaccgcaa 1981 ttctcccttg tatgtgagat cacgttcaag gtcaccctacagccgtaggc ccaggtacga 2041 cagctatgaa gcctatgagc acgaaaggct caagagggatgaataccgca aagagcacga 2101 gaagcgggag tctgaaaggg ccaaacagag agagaggcagaagcagaaag caattgaaga 2161 gcgccgtgtg atttacgttg gtaaaatcag acctgacacaacgcggacag aattgagaga 2221 ccgctttgaa gtttttggtg aaattgagga atgcaccgtaaatctgcggg atgatggaga 2281 cagctatggt ttcatcacct accgttacac ctgtgacgctttcgctgctc ttgagaatgg 2341 atatacttta cgcaggtcga acgaaactga cttcgagctgtacttttgtg gacggaagca 2401 atttttcaag tctaactatg cagacctaga taccaactcagacgattttg accctgcttc 2461 caccaagagc aagtatgact ctctggattt tgatagtttactgaaggaag ctcagagaag 2521 cttgcgcagg taacgtgttc ccaggctgag gaatgacagagagatggtca atacctcatg 2581 ggacagcgtg tcctttccca agactcttgc aagtcatacttaggaatttc tcctacttta 2641 cactctctgt acaaaaataa aacaaaacaa aacaacaataacaacaacaa caacaacaat 2701 aacaacaaca accataccag aacaagaaca acggtttacatgaacacagc tgctgaagag 2761 gcaagagaca gaatgataat ccagtaagca cacgtttattcacgggtgtc agctttgctt 2821 tccctggagg ctcttggtga cagtgtgtgt gcgtgtgtgtgtgtgggtgt gcgtgtgtgt 2881 atgtgtgtgt gtgtacttgt ttggaaagta catatgtacacatgtgagga cttgggggca 2941 cctgaacaga acgaacaagg gcgacccctt caaatggcagcatttccatg aagacacact 3001 taaaacctac aacttcaaaa tgttcgtatt ctatacaaaaggaaaataaa taaatataaa 3061 ttaaaaggaa agaaaactca caaaccaccc taaaatgacactgctgatgc ctgttgtcag 3121 cctccggtac cgtcttttca gaaagtgcaa aacccagaaagtgcaaaacc aacctgcagc 3181 aagctctctc tctctcttaa tgtaatcatt acgtgacaatcccgaagaca ctacaggttc 3241 catagaactc atatccacct ctctctctct ctctctctctctctctctct ctctctctct 3301 cctctctcct ctctcctctc tccctccctt ctttgccattgaatctgggt gggagaggat 3361 actgcaggca ccagatgcta aactttccta acattttgaagtttctgtag tttgtccttt 3421 gtcctgacac ctatgtatat gttcaaaatg ttgatcttccactgcagatt ttgaaaagcc 3481 ttgttattgg tcaagcgggg agtgtgttca gtggctccttctgaggagca gacgcggtgt 3541 tacatgagta ctgagagttg agtagaactc tctggatgtgttcagatagt gtaattgcta 3601 cattctctga tgtagttaag tatttacaga tgttaaatggagtattttta ttttatgtac 3661 atactctaca actatgttct tttttgttac agctatgcactgtaaatgca gccttctttt 3721 caaaactgct aaatttttct taatcaagaa tattcaaatgtaattatgag gtgaaacaat 3781 tattgtacac taacatattt agaagctaaa cttactgcttatatatattt gattgtaaaa 3841 aaaaaaaaaa acaaaaccaa caaaacaaaa gacagtgtgtgtgtgtgtgt ccgttgagtg 3901 caagtccaac aaaatggcgc ttcacgcaca tccatcccttcttaggtgag cttcaatcta 3961 agcatcttgt caacaacaac aaaaatccta ggcccctcaaggtattaacc acttctgcaa 4021 tatttttcca cattttcttg ttgcttgttt ttctttgaagttttatacac tggatttgtt 4081 aggggaatga aattttctca tctaaaattt ttctagacaatatcatgatt ttatgtaaag 4141 tctctcaatg gggaaccatt aagaaatgtt tttattttctctatcaacag tagatttgaa 4201 actagaggtc aaaaaaaatc tttttaaaat gctgttttgttttaattttt gtgattttaa 4261 tttgatacaa aatgctgagg taataattac agtatgatttttacaatagt caatgtgtgt 4321 ctgaagacta tctttgaagc cagtatctct ttcccttggcagagtatgat gatggtattt 4381 aatctgtatt ttttacagtt atacatcctg taaaatactgatatttcatt cctttgttta 4441 ctaaagagac atatttatca gttgcagata gcctatttattataaattaa gagatgatga 4501 aaataataag gtcagtggag actttctacc cagggtgcatggcagttgtc aggctggagt 4561 gtaccttctt cgtttgggaa actcagctct cgcagaagcagtgttccatc tttcactagc 4621 atggcctctg atacgaccat ggtgttgttc ttggtgacattgcttctgct aaatttaata 4681 ttaataataa taaatgtcag aaaaaaaacc ctccattttgagcatcagga tttcatctga 4741 gtatggagtc gctgccatgg gagtcactaa actttggagtatgtatttca tttccaaatt 4801 gagatgcatt tactgtttgg ctgacatgaa ttttctggaagatatgatag acctactact 4861 taaccgtttt tgtttgtttt tttttctttg ttgttgttgttttgtttttt gtttttttgt 4921 ttttctctct cacccaacac tatcttacaa aatgggtttcacccccaggc caatgcagct 4981 aattttgaca gctgcattca tttatcacca gcatattgtgttctgagtga atccactgtc 5041 tgtcctgtcg aatgcttgct caagtgtttg gcttattatttctaagtaga tagaaagcaa 5101 taaataacta tgaaataaaa aagaattgtg ttcacaggttctgcgttaca acagtaacac 5161 atctttaatc cgcctaattc ttgttctgta ggataaatgcaggtatttta actctttgtg 5221 aacgccaaac taaagtttac agtctttctt tctgaattttgagtatcttc tgttgtagaa 5281 taataataaa aagactatta agagcaataa attatttttaagaaatcaat atttagtaaa 5341 tcctgttatg tgtttaagga ccagatgcgt tctctattttgcctttaaat ttttgtgatc 5401 caactttaaa aacatacgtt gtcttgtttg ccctggatcatggacatgac taaaattttg 5461 tggtttcttt tcttacttat caaaagacaa cactacagatttcatgttga ggattcattg 5521 agctctcacc ctctggcctg acaaatcttg ttaccatgaagatagttttc ctccgtggac 5581 ttcaaattgc atctaaaatt agtgaagctt gtgtatcttatgcagacact gtgggtagcc 5641 catcaaaata taagctgtaa gctttgttcc tttcatttttttttttttac ttcttttggg 5701 agagaatatt tccaacaaac acatgcaccc caccaacaggggaggcaaat ttcagcatag 5761 atctataaga ctttcagatg accatgggcc attgccttcatgctgtggta agtactacat 5821 ctacaatttt ggtacccgaa ctggtgcttt agaaatgcggggtttttatt aaaaaaaaaa 5881 aaaagaaatg tagcagaata attcttttag tgcagcaactcagtttttgt aaaggactct 5941 gagaacactt gggctgtgaa cattcaaagc agcagagagggaacctggca ctattggggt 6001 aaagtgtttg ggtcagttga aaaaaaggaa accttttcatgcctttagat gtgagctaac 6061 agtaggtaat gatcatgtgt ccctttttga tggctgtacgaagaacttca atcactgtag 6121 tctaagatct gatctataga tgacctagaa tagccatgtaatataatgtg atgattctaa 6181 atttgtacct atgtgacaga cattttcaat aatgtgaaaactgcagattt gatggagcta 6241 ctttaagatt tgtaggtgaa agtgtgctac tgttggttgaactatgctga agagggaaag 6301 tgagtgatta gtttgagccc ttgctggctc ttttccacctgccaattcta catgtattgt 6361 tgtggtttta ttcattgtat gaaaattcct gtgatttttttttaaatgtg cagtacacat 6421 cagcctcact gagctaataa agggaaaaga atgtttcaaatcta

By “PGC1 beta polypeptide” is meant a protein or fragment thereof havingat least 85% homology to the sequence provided at NCBI Ref: NP_001166169or NCBI Ref: NP_573512.1 and having coactivating activity. An exemplaryhuman PGC1 beta amino acid sequence is provided below:

peroxisome proliferator-activated receptor gammacoactivator 1-beta isoform 2 [Homo sapiens]: (SEQ ID NO: 33)magndcgall deelssffln yladtqgggs geeqlyadfpeldlsqldas dfdsatcfge lqwcpenset epngyspddselfqidsene allaeltktl ddipeddvgl aafpaldggdalsctsaspa pssappspap ekpsapapev delsladstqdkkapmmqsq srsctelhkh ltsaqcclqd rglqppclqsprlpakedke pgedcpspqp apasprdsla lgradpgapvsqedmqamvq lirymhtycl pqrklppqtp eplpkacsnpsqqvrsrpws rhhskaswae fsilrellaq dvlcdvskpyrlatpvyasl tprsrprppk dsgaspgrps sveevriaaspkstgprpsl rplrlevkre vrrparlqqq eeedeeeeeeeeeeekeeee ewgrkrpgrg 1pwtklgrkl essvcpvrrsrrinpelgpw ltfadeplvp sepqgalpsl clapkaydverelgsptded sgqdqqllrg pqipalespc esgcgdmdedpscpqlpprd sprclmlals qsdptfgkks feqtltvelcgtagltpptt ppykpteedp fkpdikhslg keialslpspeglslkatpg aahklpkkhp ersellshlr hataqpasqagqkrpfscsf gdhdycqvlr pegvlqrkvl rswepsgvhledwpqqgapw aeaqapgree drscdagapp kdstllrdheirasltkhfg lletaleeed lasckspeyd tvfedsssssgessflpeee eeegeeeeed deeedsgvsp tcsdhcpyqsppskanrqlc srsrsssgss pchswspatr rnfrcesrgpcsdrtpsirh arkrrekaig egrvvyignl ssdmssrelkrrfevfgeie ecevltrnrr gekygfityr csehaalsltkgaalrkrne psfqlsyggl rhfcwprytd ydsnseealp asgkskyeam dfdsllkeaq qslh

An exemplary murine PGC1 beta polypeptide amino acid sequence isprovided below:

(SEQ ID NO: 34) MAGNDCGALLDEELSSFELNYLSDTQGGDSGEEQLCADLPELDLSQLDASDFDSATCFGELQWCPETSETEPSQYSPDDSELFQIDSENEALLAALTKTLDDIPEDDVGLAAFPELDEGDTPSCTPASPAPLSAPPSPTLERLLSPASDVDELSLLQKLLLATSSPTASSDALKDGATWSQTSLSSRSQRPCVKVDGTQDKKTPTLRAQSRPCTELHKHLTSVLPCPRVKACSPTPHPSPRLLSKEEEEEVGEDCPSPWPTPASPQDSLAQDTASPDSAQPPEEDVRAMVQLIRYMHTYCLPQRKLPQRAPEPIPQACSSLSRQVQPRSRHPPKAFWTEFSILRELLAQDILCDVSKPYRLAIPVYASLTPQSRPRPPKDSQASPAHSAMAEEVRITASPKSTGPRPSLRPLRLEVKRDVNKPTRQKREEDEEEEEEEEEEEEEKEEEEEEWGRKRPGRGLPWTKLGRKMDSSVCPVRRSRRLNPELGPWLTFTDEPLGALPSMCLDTETHNLEEDLGSLTDSSQGRQLPQGSQIPALESPCESGCGDTDEDPSCPQPTSRDSSRCLMLALSQSDSLGKKSFEESLTVELCGTAGLTPPTTPPYKPMEEDPFKPDTKLSPGQDTAPSLPSPEALPLTATPGASHKLPKRHPERSELLSHLQHATTQPVSQAGQKRPFSCSFGDHDYCQVLRPEAALQRKVLRSWEPIGVHLEDLAQQGAPLPTETKAPRREANQNCDPTHKDSMQLRDHEIRASLTKHEGLLETALEGEDLASCKSPEYDTVFEDSSSSSGESSFLLEEEEEEEEGGEEDDEGEDSGVSPPCSDHCPYQSPPSKASRQLCSRSRSSSGSSSCSSWSPATRKNERRESRGPCSDGTPSVRHARKRREKAIGEGRVVYIRNLSSDMSSRELKKRFEVEGEIVECQVLTRSKRGQKHGFITERCSEHAALSVRNGATLRKRNEPSFHLSYGGLRHFRWPRYTDYDPTSEESLPSSGKSKYEAMDFDSLLKEAQQSLH

By “PGC1 beta polynucleotide” is meant a nucleic acid molecule encodinga PGC1 beta polypeptide. An exemplary human PGC1 beta polynucleotidesequence is provided at NM_001172698:

(SEQ ID NO: 35) 1ctcctccctc ctcccttgct cgctcgctgg ctccctcccc ccgggccggc tcggcgttga 61ctccgccgca cgctgcagcc gcggctggaa gatggcgggg aacgactgcg gcgcgctgct 121ggacgaagag ctctcctcct tcttcctcaa ctatctcgct gacacgcagg gtggagggtc 181cggggaggag caactctatg ctgactttcc agaacttgac ctctcccagc tggatgccag 241cgactttgac tcggccacct gctttgggga gctgcagtgg tgcccagaga actcagagac 301tgaacccaac cagtacagcc ccgatgactc cgagctcttc cagattgaca gtgagaatga 361ggccctcctg gcagagctca ccaagaccct ggatgacatc cctgaagatg acgtgggtct 421ggctgccttc ccagccctgg atggtggaga cgctctatca tgcacctcag cttcgcctgc 481cccctcatct gcacccccca gccctgcccc ggagaagccc tcggccccag cccctgaggt 541ggacgagctc tcactggcgg acagcaccca agacaagaag gctcccatga tgcagtctca 601gagccgaagt tgtacagaac tacataagca cctcacctcg gcacagtgct gcctgcagga 661tcggggtctg cagccaccat gcctccagag tccccggctc cctgccaagg aggacaagga 721gccgggtgag gactgcccga gcccccagcc agctccagcc tctccccggg actccctagc 781tctgggcagg gcagaccccg gtgccccggt ttcccaggaa gacatgcagg cgatggtgca 841actcatacgc tacatgcaca cctactgcct cccccagagg aagctgcccc cacagacccc 901tgagccactc cccaaggcct gcagcaaccc ctcccagcag gtcagatccc ggccctggtc 961ccggcaccac tccaaagcct cctgggctga gttctccatt ctgagggaac ttctggctca 1021agacgtgctc tgtgatgtca gcaaacccta ccgtctggcc acgcctgttt atgcctccct 1081cacacctcgg tcaaggccca ggccccccaa agacagtcag gcctcccctg gtcgcccgtc 1141ctcggtggag gaggtaagga tcgcagcttc acccaagagc accgggccca gaccaagcct 1201gcgcccactg cggctggagg tgaaaaggga ggtccgccgg cctgccagac tgcagcagca 1261ggaggaggaa gacgaggaag aagaggagga ggaagaggaa gaagaaaaag aggaggagga 1321ggagtggggc aggaaaaggc caggccgagg cctgccatgg acgaagctgg ggaggaagct 1381ggagagctct gtgtgccccg tgcggcgttc tcggagactg aaccctgagc tgggcccctg 1441gctgacattt gcagatgagc cgctggtccc ctcggagccc caaggtgctc tgccctcact 1501gtgcctggct cccaaggcct acgacgtaga gcgggagctg ggcagcccca cggacgagga 1561cagtggccaa gaccagcagc tcctacgggg accccagatc cctgccctgg agagcccctg 1621tgagagtggg tgtggggaca tggatgagga ccccagctgc ccgcagctcc ctcccagaga 1681ctctcccagg tgcctcatgc tggccttgtc acaaagcgac ccaacttttg gcaagaagag 1741ctttgagcag accttgacag tggagctctg tggcacagca ggactcaccc cacccaccac 1801accaccgtac aagcccacag aggaggatcc cttcaaacca gacatcaagc atagtctagg 1861caaagaaata gctctcagcc tcccctcccc tgagggcctc tcactcaagg ccaccccagg 1921ggctgcccac aagctgccaa agaagcaccc agagcgaagt gagctcctgt cccacctgcg 1981acatgccaca gcccagccag cctcccaggc tggccagaag cgtcccttct cctgttcctt 2041tggagaccat gactactgcc aggtgctccg accagaaggc gtcctgcaaa ggaaggtgct 2101gaggtcctgg gagccgtctg gggttcacct tgaggactgg ccccagcagg gtgccccttg 2161ggctgaggca caggcccctg gcagggagga agacagaagc tgtgatgctg gcgccccacc 2221caaggacagc acgctgctga gagaccatga gatccgtgcc agcctcacca aacactttgg 2281gctgctggag accgccctgg aggaggaaga cctggcctcc tgcaagagcc ctgagtatga 2341cactgtcttt gaagacagca gcagcagcag cggcgagagc agcttcctcc cagaggagga 2401agaggaagaa ggggaggagg aggaggagga cgatgaagaa gaggactcag gggtcagccc 2461cacttgctct gaccactgcc cctaccagag cccaccaagc aaggccaacc ggcagctctg 2521ttcccgcagc cgctcaagct ctggctcttc accctgccac tcctggtcac cagccactcg 2581aaggaacttc agatgtgaga gcagagggcc gtgttcagac agaacgccaa gcatccggca 2641cgccaggaag cggcgggaaa aggccattgg ggaaggccgc gtggtgtaca ttcaaaatct 2701ctccagcgac atgagctccc gagagctgaa gaggcgcttt gaagtgtttg gtgagattga 2761ggagtgcgag gtgctgacaa gaaataggag aggcgagaag tacggcttca tcacctaccg 2821gtgttctgag cacgcggccc tctctttgac aaagggcgct gccctgagga agcgcaacga 2881gccctccttc cagctgagct acggagggct ccggcacttc tgctggccca gatacactga 2941ctacgattcc aattcagaag aggcccttcc tgcgtcaggg aaaagcaagt atgaagccat 3001ggattttgac agcttactga aagaggccca gcagagcctg cattgataac agccttaacc 3061ctcgaggaat acctcaatac ctcagacaag gcccttccaa tatgtttacg ttttcaaaga 3121aatcaagtat atgaggagag cgagcgagcg tgagagaaca cccgtgagag agacttgaaa 3181ctgctgtcct ttaaaaaaaa aaaaaatcaa tgtttacatt gaacaaagct gcttctgtct 3241gtgagtttcc atggtgttga cgttccactg ccacattagt gtcctcgctt ccaacgggtt 3301gtcccgggtg cacctcgaag tgccgggtcc gtcacccatc gccccttcct tcccgactga 3361cttcctctcg tagacttgca gctgtgttca ccataacatt tcttgtctgt agtgtgtgat 3421gatgaaattg ttacttgtga atagaatcag gactataaac ttcattttta attgaaaaaa 3481aaagtatatc cttaaaataa tgtatttatg gctcagatgt actgtgcctg ggattattgt 3541attgcttcct tgatttttta actatgcact gtcatgaggt gtttgccact gagctgccct 3601gctccccttg ccagattgcc ctggaggtgc tgggtggccg ctaggctggt ctgcaggaaa 3661gcgcggcctg ccgtttccgg gccgtatctg ccaagccctg ccttgtctct tactgagcaa 3721gtttggctca aattatagga gcccccatct tgtgcccagc tcatgctcca agtgtgtgtc 3781tatccatttg tactcagact cttgagtacc ttgtaaggaa ggcggggcaa gctgcatcat 3841tcctgttttc caggggaggc tggcagctcc tcaagaggcg aaatgactgt gggaggtccg 3901gttaccagtg aggaggcaga gcggtgaccc agaccaggcc ttctggttct tggtcccgtg 3961cttccgtagt agctggggta aagacaccgt ttcagggact ggtagaggtg agttcggcta 4021aattgggcac cgggctagaa gcctaagggc tcattttagg ggttacatta ggtgttgatt 4081caccagcatc aggtgaattc aagccctggc atgtgtcttg gatgcaccat cagctttgat 4141cctgagtggt cctgcggttt gtctgtgcct gtggacacac tgtcagaact tcagtgacac 4201ccctggcagc ggtacagaca ggtggtctgg gagcagtcat cttttttggg ccagccacca 4261gcccatccta ctccctcagg tagtccttcg tctttacctt gtccttgtct gtaaagttgt 4321tttggtggct ggggcagggg agccaggagg agggagtgaa ggttgggaat agataggaca 4381atctcctagc tctcctccaa ttgagaaaac actccaattg ggctttgctt taaactttgt 4441gttcttaagt gatgtcaaag ccatttccag cttaatgttc tgtgggtacc ttgggggcca 4501ttcatgcagg gagcatggcc aggcagggta tgagtacatt gtttctgatt tctttcatac 4561atcagggttc ctcgggaaat ttttgtattt tttttttaag tcctgctgct ttaaaaattt 4621gaaagtggct cattaaacta aacaggctaa tgtaatttgt tgcttatgcc aagcctagac 4681tgttgagaat tgacgttttt aaagattatc aaatacctca gtaggtaaaa tgagcccatg 4741atcttccact gagtggtgag catactccca gcccatggac aaggccggaa gagacaggct 4801ttagtagggg tagggaattt gaactgttgt gtgtcacagc agttgacctc tctggactcc 4861aatttccttt cctgtgaaat gaactgatta gacatgtttc aacattgtta gcttctgctg 4921aggcagtgtc tagcccaaga tggcaaatac atagctcatg tgccactact cccacctcct 4981tgaccaatac agacataact aatcaatcac accactcagg ttccctgagc ctggatgtgc 5041tataagaatc ctgaaatcag tgctctggta agtcattact aattgattag agttcaatct 5101atttgacatc ttgggctaat ctttggaagg tttccaacaa tcacacaaaa ccatatgctg 5161gctgggtttc atgctggcct atccctgtct gtgatgttcc gttccatgag agaaaactcc 5221cctaatgcta ttccatggcg taacactccc aatactattt tgacgcccac gtccccttgc 5281agagggtgca gggggcggta gacgaatgac agacaggaac atatttgggg aaggcagggc 5341ttaggaagat ggaccaaaaa gggacttccc acagcacaga cctgatcatt cggatttcct 5401ctttagctat tcactgccta gcacatagta ggcacacaat aaatgattat ggaatgggat 5461aaaatttaga tctttctgct gcctccacta agttaagtcc tgatttacat caaggagaga 5521actgagatag gaaagaacac tagattccaa gtctggagag ttgggggagt ccagattcta 5581ccaagaattt cctttgtaac tttggtaagt cccttttact ccctggcacc ccggtgtgct 5641gaaaggagtt ggtccatata tgatctctta gcccctccta tttgcttctt ccttgattgc 5701tcttggtcaa agggtcagcc ttgggctggt gatactttag agtaaagaaa tggagagttt 5761tagcaaagga ccagtctgtc cctccctgct ttggggtcag ctaaagctgt cctttcatgt 5821cagattaacc taggacactt gtagttagct tagacgttgg cccttgagca gagacctgag 5881cgtggcattg ggacatgaca tacctaaagt cagggctagg ggacgctgcc tgccaagggc 5941atcgagtagt ctctacttgc tatcccgtac ataaaatgct acaagttcta aaatttaccg 6001accctgcaga caacctctat cccgaaggac tcattcggtg ctgtgtatta tttagggcaa 6061ctccaaggtc tattcagaaa aacgagtgaa ccttggtctc tttcccacca aattgaggag 6121taacccagag ggagcagctg ccattggcaa ccatctcgtt gtagctctgt cctagtgttt 6181gctcttgatg atgtttacat gtgatcgcca taaagcttgc tgtagactgt gtcgatagcc 6241gcccgcacag ggcaggtcgt actgtccgtt tctgtgccgt gctggtgttt tccaaaaatg 6301tctgatccaa ccactaagtg gaattcttcc atctccttcc tcagtctgta caaggctgaa 6361tcagaatccc cattctcggg ggctctggtt accgaaggaa aatgcatcaa agagttaaag 6421aatatgagtg gatggagtgc agctaaggcc cccaccccct gctccgtcac aacttgcccc 6481ctcaaccaaa aagctgcttt gagtcaaaaa gcacccataa gatacctgca tctgccttga 6541aatcttgcag catggagtgt catatgtact caggagagag gcagggcttt gcgggcagga 6601gaaggaaggg aggaatgctc tgagctgcaa agacccagta ctcaagttct gacgtgggag 6661gagatgcagt gagacgtctc ttgttgccta aagcctgttc ctgttggttt tcttagagtg 6721atttctccta gacatgtgca gtaggcccac tggggctgct gtgcagtggt gagtaaaagg 6781gcagggaagg catggacagc ctggtccttc tgcatggaca gctcagtcca tggcccatcc 6841caggtataga gttcagttaa tcccatttga gcctgcagct taagagatgg ctcatcctaa 6901ctgtgaagca aaatcagccc cagaggatgt attgatctga ctcactgatg tcaaaattgc 6961agtatttttt tagcatttga gatttagcag ctgccttcag tttggggtta cccacatccc 7021agcatcagat atgattaagg aaagaaattg gatgtacaac agcaaagaaa gtgaatgtca 7081tggtttccct ggccaaagaa gagggaccct gtcatcctta ccaatgggga agaagaaaac 7141tagtgcatgt gcaatatgtc aaagttagtc ccctagtccc tgaggggttt ttacacacag 7201atgggctcca ggtctgctcg tcaagtttgg aggtaccggg taaatggagg ggagctgcag 7261agttggaaac ccacatgcat ggatgtgtcc ttggcccaga accaccatgg gatgggggag 7321gccctgagcc ggctacaaga cacccaggaa gtaggcaaag gctgactttg cattaaacaa 7381taaaagcact ttgagaaaac cccaacactt cagcctgggt ccgtgtttct acactggaaa 7441atacgagtct cctttggctg tgtgaagtga tcttctagag actgggacag ggagtttggg 7501aatggggctg ctgtcaggta ggagagagca gagatgcctt tggagatgtc agcagcagga 7561gagccagtgc tggggccaac cctttgctgg ccttttgttg gaagcccttg aaacagggag 7621ccatgggttt agatcttggt acctaccttt acagaaagat gaaaacagcc cagctgagtg 7681aaatgagttt gtagagtaag tcacttaact gtaagccatc tcagaatcag aaaccctaat 7741gtttcttact tgctatgtga ccttgggccc ctgtttcctc atctaccaaa tgagaatgtt 7801gaatatgagc attaaagtcc ctttcacctc tgagaggctc agatccccaa ccaggagcat 7861tgggaatcca tcactcctcc ttgaaactga ttccattctc tgacttgacc cagctcctgt 7921tcagggtgag ggttctctgc aagaaccaac cagcagtagg ttcaatccca ctgtgtcctg 7981gctgagttgc cttatccaag aagaccagct ccccgggaca gatctaagcc atagtttcta 8041gtggggacag taaggaatta aacccccaac ttggctaggt aacgatgtca aatctcacat 8101taaccttgtc tttgtcccca ctggatagct gttaatccga atgttgtgac catttggctg 8161tttctctctt gttctcagac aatactagca atacactttt tttttttttt tttaaagaaa 8221aacagcttag gagcttttca cacatttctt tcaaatgatt gtaaaacata tggggcaaca 8281ggaggcattg atcgcgctgc atatgtttag ggcagctttt gttttttgtt tctttaatgg 8341tatagcagca gtgactgagc cttcgtgatt cctggggaca gcttttcaga tactctgttt 8401catcagtatg ctttgcacat ccggaaggag tacaaaaatc caactgccca aatttggggc 8461ttggaaaata ggttttatag gtggtcggtc cctgggctgt gcaacaactc ctcaaagagg 8521ggtttatata actagaaccc ccctgggctg tatttttggt caaaggagtc tccaaggcgg 8581cttacaaaag cttccttttt cacttgacca cccttgctca ttggttactt gtgaagggaa 8641ttggtcagtt tccacctcag cactttgcct tatcaacatg cggtcgccat ctagtggcca 8701aaggttgtct ccaccagcta cccagatgga aggcaaataa atcctttcgg ccaccctgct 8761gtccatcgtg aactttggga atgaaatata atggcctgaa cgaactgcct ttgtgttcag 8821agatcagtgc aacactaggg tcagaagact ccagaagcag ccacttagta gactctcacg 8881cagaactgag aaatgcacta gctgtcctgt gggcagaaga gacaggagtg gaccaggaga 8941ggtccaggtg cccgggaagg gtttactgta actgcaatac tggcagccca gctgctgacc 9001ttgttaagta aacctttgct gggtggtccg aattctgccc tcaaggcaag ataagaagtt 9061gggtgtaagg attttgtggg gggcctggcc atgatctttg atatgatccc cgaatagcca 9121aatagttttt tttgttcaat tttttgtttc tgtattttgt atttttaaaa tcttgtcaaa 9181tgtttttgtg ttaggaataa aaagtcataa actattccca actttgtttc ttgagggatg 9241ttctgattcc aatggaaaca ggtgggaaat ctcaagggga gcgtggacaa ggtggtatgt 9301gcagcagggg aatagactgc ttggatttcc aaatggtttc tggggaagat gaccatccag 9361aagtccagct tagtgcagtc tgctctggaa ttcacaccca ccccctcgcc tccttgtgcc 9421atgttgttag cattggcttg gagcatctgc ttcttccaga ggcagctgct aatgttgaaa 9481ccaacacgag ccctctcccc aaccccaggt ttctaaagaa ggtgtctgta gccagcctta 9541atcaactggg caaggtggtc cctatggtcc tttccagcat ttccaaatct tggactcaaa 9601ttattttctc ttggtgtgac cacacagcct agagaattct gagcaatagg agccagggct 9661ttccctgact ctgcgacagg gtcaaaccaa ggaatggcta aacctgtgag gttttgtcat 9721ccccgggggt actactgtag ggggcattat ttattaggaa gcttaacaag gtaactacgg 9781cctgagtgcg tgagtgtaag gctgtgtttg tggtgggggt gtgtgtgtgt gtatctgtgc 9841acacatacac acgtctgtgc ctgtgtgtgt gtgtttgtgt gtgtgtgtgt gtgtggaatt 9901acattgatgc atttattgag aaaggtgcaa gaatttcacc tacacagagg gacacatctg 9961ctttgttatt tataatagaa agctaaattt taatttttta aaggacactg ctaatgattg 10021agaatcaagt ttttagtttt gctatttttt ttaattggta gaggattttt atatattttt 10081tccattttgt tgggttgtgt ccttatttat ataaatactt tatccgtaag aggcaaggag 10141gaaaccttct ttgcttttac atattgtggt tgtcatcgtc cctattttat ttctggtgtg 10201atttctctgt cttaccttct aaatgagaaa atgttttctt gtatttgtac attgtcagat 10261tctatagttt cctagataat ttaaccaaat tgctctatgt attattattc tgtgagtata 10321aagttctatt ttaatgtctg taaatacttc agaactggct tcttttctca aactcccact 10381gtggggttat tgtttacatc acagaaactg tagaatctct atgctcatgt actgtaaata 10441gtgaagtgat ctgcttataa ataaacttaa caaatacact atggagatta aaaacaaaat 10501accacccaca aaaaaaaaaa aaaaa

An exemplary murine PGC1 beta polynucleotide sequence is provided atNM_133249.2:

(SEQ ID NO: 36) 1 ctcgctccct cccccgggcg ggctcggcgc tgactccgccgcacgctgca gccgcggctg 61 gaagatggcg gggaacgact gcggcgcgct gctggatgaagagctctcgt ccttcttcct 121 caactatctc tctgacacgc agggtgggga ctctggagaggaacagctgt gtgctgactt 181 gccagagctt gacctctccc agctggacgc cagtgactttgactcagcca cgtgctttgg 241 ggagctgcag tggtgcccgg agacctcaga gacagagcccagccagtaca gccccgatga 301 ctccgagctc ttccagattg acagtgagaa tgaagctctcttggctgcgc ttacgaagac 361 cctggatgac atccccgaag acgatgtggg gctggctgccttcccagaac tggatgaagg 421 cgacacacca tcctgcaccc cagcctcacc tgcccccttatctgcacccc ccagccccac 481 cctggagagg cttctgtccc cagcgtctga cgtggacgagctttcactgc tacagaagct 541 cctcctggcc acatcctccc caacagcaag ctctgacgctctgaaggacg gggccacctg 601 gtcccagacc agcctcagtt ccagaagtca gcggccttgtgtcaaggtgg atggcaccca 661 ggataagaag acccccacac tgcgggctca gagccggccttgtacggaac tgcataagca 721 cctcacttcg gtgctgccct gtcccagagt gaaagcctgctccccaactc cgcacccgag 781 ccctcggctc ctctccaaag aggaggagga ggaggtgggggaggattgcc caagcccttg 841 gccgactcca gcctcgcccc aagactccct agcacaggacacggccagcc ccgacagtgc 901 ccagcctccc gaggaggatg tgagggccat ggtacagctcattcgctaca tgcataccta 961 ctgcctgcct cagaggaagc tgccccaacg ggccccagagccaatccccc aggcctgcag 1021 cagcctctcc aggcaggttc aaccccgatc ccggcatccccccaaagcct tctggactga 1081 gttctctatc ctaagggaac ttctggccca agatatcctctgtgatgtta gcaagcccta 1141 ccgcctggcc atacctgtct atgcttccct cacacctcagtccaggccca ggccccccaa 1201 ggacagtcag gcctcccctg cccactctgc catggcagaagaggtgagaa tcactgcttc 1261 ccccaagagc accgggccta gacccagcct gcgtcctctgaggctggagg tgaaacggga 1321 tgttaacaag cctacaaggc aaaagcggga ggaagatgaggaggaggagg aggaagaaga 1381 agaagaggaa gaagaaaaag aagaggaaga agaggagtggggcaggaaga gaccaggtcg 1441 tggcctgcca tggaccaaac tagggaggaa gatggacagctccgtgtgcc ccgtgcggcg 1501 ctccaggaga ctgaatccag agctgggtcc ctggctgacattcactgatg agcccttagg 1561 tgctctgccc tcgatgtgcc tggatacaga gacccacaacctggaggaag acctgggcag 1621 cctcacagac agtagtcaag gccggcagct cccccagggatcccagatcc ccgccctgga 1681 aagcccctgt gagagtgggt gcggagacac agatgaagatccaagctgcc cacagcccac 1741 ttccagagac tcctccaggt gcctcatgct ggccttgtcacaaagcgact ctcttggcaa 1801 gaagagcttt gaggagtccc tgacggtgga gctttgcggcacggcaggac tcacgccacc 1861 caccacacct ccatacaagc caatggagga ggaccccttcaagccagaca ccaagctcag 1921 cccaggccaa gacacagctc ccagccttcc ctcccccgaggctcttccgc tcacagccac 1981 cccaggagct tcccacaagc tgcccaagag gcacccagagcgaagcgagc tcctgtccca 2041 tttgcagcat gccacaaccc aaccagtctc acaggctggccagaagcgcc ccttctcctg 2101 ctcctttgga gaccacgact actgccaggt gctcaggccagaggctgccc tgcagaggaa 2161 ggtgctgcgg tcctgggagc caatcggggt ccaccttgaagacttggccc agcagggtgc 2221 ccctctgcca acggaaacaa aggcccctag gagggaggcaaaccagaact gtgaccctac 2281 ccacaaggac agcatgcagc taagagacca tgagatccgtgccagtctca caaagcactt 2341 tgggctgctg gagactgctc tggaaggtga agacctggcgtcctgtaaaa gcccggagta 2401 tgacaccgta tttgaggaca gcagcagcag cagtggcgagagtagcttcc tgcttgagga 2461 ggaggaggaa gaggaggagg gaggggaaga ggacgatgaaggagaggact caggggtcag 2521 ccctccctgc tctgatcact gcccctacca gagcccacccagtaaggcca gtcggcagct 2581 ctgctcccga agccgctcca gttccggctc ctcgtcctgcagctcctggt caccagccac 2641 ccggaagaac ttcagacgtg agagcagagg gccctgttcagatggaaccc caagcgtccg 2701 gcatgccagg aagcggcggg aaaaggccat cggtgaaggccgtgtggtat acattcgaaa 2761 tctctccagt gacatgagct ctcgggaact aaagaagcgctttgaggtgt tcggtgagat 2821 tgtagagtgc caggtgctga cgagaagtaa aagaggccagaagcacggtt ttatcacctt 2881 ccggtgttca gagcacgctg ccctgtccgt gaggaacggcgccaccctga gaaagcgcaa 2941 tgagccctcc ttccacctga gctatggagg gctccggcacttccgttggc ccagatacac 3001 tgactatgat cccacatctg aggagtccct tccctcatctgggaaaagca agtacgaagc 3061 catggatttt gacagcttac tgaaagaggc ccagcagagcctgcattgat atcagcctta 3121 accttcgagg aatacctcaa tacctcagac aaggcccttccaatatgttt acgttttcaa 3181 agaaaagagt atatgagaag gagagcgagc gagcgagcgagcgagcgagt gagcgtgaga 3241 gatcacacag gagagagaaa gacttgaatc tgctgtcgtttcctttaaaa aaaaaaaaac 3301 gaaaaacaaa aacaaatcaa tgtttacatt gaacaaagctgcttccgtcc gtctgtccgt 3361 ccgtccgtcc gtccgtgagt ttccatgctg ttgatgttccactgccacgt tagcgtcgtc 3421 ctcgcttcca gcggatcgtc ctgggtgcgc ctccaagtgctgtcagtcgt cctctgcccc 3481 tcccacccga ctgacttcct tctgttagac ttgagctgtgttcacataac atcttctgtc 3541 tgtagagtgt gatgatgaca ttgttacttg tgaatagaatcaggagttag aaactcattt 3601 ttaattgaag aaaaaaaaag tatatcctta aaaagaaaaaaaaaaaaaca aatgta

By “operably linked” is meant that a first polynucleotide is positionedadjacent to a second polynucleotide that directs transcription of thefirst polynucleotide when appropriate molecules (e.g., transcriptionalactivator proteins) are bound to the second polynucleotide.

By “positive” is meant that a cell expresses a detectable level of amarker.

By “promoter” is meant a polynucleotide sufficient to directtranscription.

By “reference” is meant a standard or control condition. In oneembodiment, a reference cell is a cell that expresses Sca1 and/or CD34.In another embodiment, the reference cell expresses Sca1 and/or CD34 andalso expresses Oct4, Sox2, Klf4 and cMyc (OSKM).

A “reference sequence” is a defined sequence used as a basis forsequence comparison. A reference sequence may be a subset of or theentirety of a specified sequence; for example, a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence. For polypeptides, the length of the reference polypeptidesequence will generally be at least about 16 amino acids, preferably atleast about 20 amino acids, more preferably at least about 25 aminoacids, and even more preferably about 35 amino acids, about 50 aminoacids, or about 100 amino acids. For nucleic acids, the length of thereference nucleic acid sequence will generally be at least about 50nucleotides, preferably at least about 60 nucleotides, more preferablyat least about 75 nucleotides, and even more preferably about 100nucleotides or about 300 nucleotides or any integer thereabout ortherebetween.

By “reprogramming” is meant altering a cell such that at least oneprotein product is produced in the reprogrammed cell that is notproduced in the cell prior to reprogramming or that is not expressed ina corresponding control cell. Typically, the reprogrammed cell has analtered transcriptional or translational profile, such that thereprogrammed cell expresses a set of proteins not expressed in the cellprior to reprogramming (or in a corresponding control cell).

By “regenerate” is meant capable of contributing at least one cell tothe repair or de novo construction of a tissue or organ.

Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule.Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences (e.g., a gene described herein),or portions thereof, under various conditions of stringency. (See, e.g.,Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and more preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and more preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred: embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/mldenatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variationson these conditions will be readily apparent to those skilled in theart.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

By “SOX2 polypeptide” is meant a protein or fragment thereof having atleast 85% homology to the sequence provided at NCBI Ref: NP_003097.1(human) or NP_035573.3 (murine). An exemplary human amino acid sequenceis provided below:

(SEQ ID NO: 37) MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYSMMQDQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMISMYLPGAEVPEPAAPSRLHMSQHYQSGPVPGT AINGTLPLSHM

An exemplary murine amino acid sequence is provided below:

(SEQ ID NO: 38) MYNMMETELKPPGPQQASGGGGGGGNATAAATGGNQKNSPDRVKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYSMMQDQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMISMYLPGAEVPEPAAPSRLHMSQHY QSGPVPGTAINGTLPLSHM

By “SOX2 polynucleotide” is meant a nucleic acid molecule encoding aSOX2 polypeptide. An exemplary human SOX2 polynucleotide sequence isprovided at NM_003106:

(SEQ ID NO: 39) 1 ggatggttgt ctattaactt gttcaaaaaa gtatcaggagttgtcaaggc agagaagaga 61 gtgtttgcaa aagggggaaa gtagtttgct gcctctttaagactaggact gagagaaaga 121 agaggagaga gaaagaaagg gagagaagtt tgagccccaggcttaagcct ttccaaaaaa 181 taataataac aatcatcggc ggcggcagga tcggccagaggaggagggaa gcgctttttt 241 tgatcctgat tccagtttgc ctctctcttt ttttcccccaaattattctt cgcctgattt 301 tcctcgcgga gccctgcgct cccgacaccc ccgcccgcctcccctcctcc tctccccccg 361 cccgcgggcc ccccaaagtc ccggccgggc cgagggtcggcggccgccgg cgggccgggc 421 ccgcgcacag cgcccgcatg tacaacatga tggagacggagctgaagccg ccgggcccgc 481 agcaaacttc ggggggcggc ggcggcaact ccaccgcggcggcggccggc ggcaaccaga 541 aaaacagccc ggaccgcgtc aagcggccca tgaatgccttcatggtgtgg tcccgcgggc 601 agcggcgcaa gatggcccag gagaacccca agatgcacaactcggagatc agcaagcgcc 661 tgggcgccga gtggaaactt ttgtcggaga cggagaagcggccgttcatc gacgaggcta 721 agcggctgcg agcgctgcac atgaaggagc acccggattataaataccgg ccccggcgga 781 aaaccaagac gctcatgaag aaggataagt acacgctgcccggcgggctg ctggcccccg 841 gcggcaatag catggcgagc ggggtcgggg tgggcgccggcctgggcgcg ggcgtgaacc 901 agcgcatgga cagttacgcg cacatgaacg gctggagcaacggcagctac agcatgatgc 961 aggaccagct gggctacccg cagcacccgg gcctcaatgcgcacggcgca gcgcagatgc 1021 agcccatgca ccgctacgac gtgagcgccc tgcagtacaactccatgacc agctcgcaga 1081 cctacatgaa cggctcgccc acctacagca tgtcctactcgcagcagggc acccctggca 1141 tggctcttgg ctccatgggt tcggtggtca agtccgaggccagctccagc ccccctgtgg 1201 ttacctcttc ctcccactcc agggcgccct gccaggccggggacctccgg gacatgatca 1261 gcatgtatct ccccggcgcc gaggtgccgg aacccgccgcccccagcaga cttcacatgt 1321 cccagcacta ccagagcggc ccggtgcccg gcacggccattaacggcaca ctgcccctct 1381 cacacatgtg agggccggac agcgaactgg aggggggagaaattttcaaa gaaaaacgag 1441 ggaaatggga ggggtgcaaa agaggagagt aagaaacagcatggagaaaa cccggtacgc 1501 tcaaaaagaa aaaggaaaaa aaaaaatccc atcacccacagcaaatgaca gctgcaaaag 1561 agaacaccaa tcccatccac actcacgcaa aaaccgcgatgccgacaaga aaacttttat 1621 gagagagatc ctggacttct ttttggggga ctatttttgtacagagaaaa cctggggagg 1681 gtggggaggg cgggggaatg gaccttgtat agatctggaggaaagaaagc tacgaaaaac 1741 tttttaaaag ttctagtggt acggtaggag ctttgcaggaagtttgcaaa agtctttacc 1801 aataatattt agagctagtc tccaagcgac gaaaaaaatgttttaatatt tgcaagcaac 1861 ttttgtacag tatttatcga gataaacatg gcaatcaaaatgtccattgt ttataagctg 1921 agaatttgcc aatatttttc aaggagaggc ttcttgctgaattttgattc tgcagctgaa 1981 atttaggaca gttgcaaacg tgaaaagaag aaaattattcaaatttggac attttaattg 2041 tttaaaaatt gtacaaaagg aaaaaattag aataagtactggcgaaccat ctctgtggtc 2101 ttgtttaaaa agggcaaaag ttttagactg tactaaattttataacttac tgttaaaagc 2161 aaaaatggcc atgcaggttg acaccgttgg taatttataatagcttttgt tcgatcccaa 2221 ctttccattt tgttcagata aaaaaaacca tgaaattactgtgtttgaaa tattttctta 2281 tggtttgtaa tatttctgta aatttattgt gatattttaaggttttcccc cctttatttt 2341 ccgtagttgt attttaaaag attcggctct gtattatttgaatcagtctg ccgagaatcc 2401 atgtatatat ttgaactaat atcatcctta taacaggtacattttcaact taagttttta 2461 ctccattatg cacagtttga gataaataaa tttttgaaatatggacactg aaaaaaaaaa

An exemplary murine SOX2 polynucleotide sequence is provided atNM_011443.3:

(SEQ ID NO: 40) 1 ctattaactt gttcaaaaaa gtatcaggag ttgtcaaggcagagaagaga gtgtttgcaa 61 aaagggaaaa gtactttgct gcctctttaa gactagggctgggagaaaga agaggagaga 121 gaaagaaagg agagaagttt ggagcccgag gcttaagcctttccaaaaac taatcacaac 181 aatcgcggcg gcccgaggag gagagcgcct gttttttcatcccaattgca cttcgcccgt 241 ctcgagctcc gcttcccccc aactattctc cgccagatctccgcgcaggg ccgtgcacgc 301 cgaggccccc gcccgcggcc cctgcatccc ggcccccgagcgcggccccc acagtcccgg 361 ccgggccgag ggttggcggc cgccggcggg ccgcgcccgcccagcgcccg catgtataac 421 atgatggaga cggagctgaa gccgccgggc ccgcagcaagcttcgggggg cggcggcgga 481 ggaggcaacg ccacggcggc ggcgaccggc ggcaaccagaagaacagccc ggaccgcgtc 541 aagaggccca tgaacgcctt catggtatgg tcccgggggcagcggcgtaa gatggcccag 601 gagaacccca agatgcacaa ctcggagatc agcaagcgcctgggcgcgga gtggaaactt 661 ttgtccgaga ccgagaagcg gccgttcatc gacgaggccaagcggctgcg cgctctgcac 721 atgaaggagc acccggatta taaataccgg ccgcggcggaaaaccaagac gctcatgaag 781 aaggataagt acacgcttcc cggaggcttg ctggcccccggcgggaacag catggcgagc 841 ggggttgggg tgggcgccgg cctgggtgcg ggcgtgaaccagcgcatgga cagctacgcg 901 cacatgaacg gctggagcaa cggcagctac agcatgatgcaggagcagct gggctacccg 961 cagcacccgg gcctcaacgc tcacggcgcg gcacagatgcaaccgatgca ccgctacgac 1021 gtcagcgccc tgcagtacaa ctccatgacc agctcgcagacctacatgaa cggctcgccc 1081 acctacagca tgtcctactc gcagcagggc acccccggtatggcgctggg ctccatgggc 1141 tctgtggtca agtccgaggc cagctccagc ccccccgtggttacctcttc ctcccactcc 1201 agggcgccct gccaggccgg ggacctccgg gacatgatcagcatgtacct ccccggcgcc 1261 gaggtgccgg agcccgctgc gcccagtaga ctgcacatggcccagcacta ccagagcggc 1321 ccggtgcccg gcacggccat taacggcaca ctgcccctgtcgcacatgtg agggctggac 1381 tgcgaactgg agaaggggag agattttcaa agagatacaagggaattggg aggggtgcaa 1441 aaagaggaga gtaggaaaaa tctgataatg ctcaaaaggaaaaaaaatct ccgcagcgaa 1501 acgacagctg cggaaaaaaa ccaccaatcc catccaaattaacgcaaaaa ccgtgatgcc 1561 gactagaaaa cttttatgag agatcttggg acttctttttgggggactat ttttgtacag 1621 agaaaacctg agggcggcgg ggagggcggg ggaatcggaccatgtataga tctggaggaa 1681 aaaaactacg caaaactttt ttttaaagtt ctagtggtacgttaggcgct tcgcagggag 1741 ttcgcaaaag tctttaccag taatatttag agctagactccgggcgatga aaaaaaagtt 1801 ttaatatttg caagcaactt ttgtacagta tttatcgagataaacatggc aatcaaatgt 1861 ccattgttta taagctgaga atttgccaat atttttcgaggaaagggttc ttgctgggtt 1921 ttgattctgc agcttaaatt taggaccgtt acaaacaaggaaggagttta ttcggatttg 1981 aacattttag ttttaaaatt gtacaaaagg aaaacatgagagcaagtact ggcaagaccg 2041 ttttcgtggt cttgtttaag gcaaacgttc tagattgtactaaattttta acttactgtt 2101 aaaggcaaaa aaaaaatgtc catgcaggtt gatatcgttggtaatttata atagcttttg 2161 ttcaatccta ccctttcatt ttgttcacat aaaaaatatggaattactgt gtttgaaata 2221 ttttcttatg gtttgtaata tttctgtaaa ttgtgatattttaaggtttt tccccccttt 2281 tattttccgt agttgtattt taaaagattc ggctctgttattggaatcag gctgccgaga 2341 atccatgtat atatttgaac taataccatc cttataacagctacattttc aacttaagtt 2401 tttactccat tatgcacagt ttgagataaa taaatttttgaaatatggac actgaaa

By “IDH3α polypeptide” is meant a protein or fragment thereof having atleast 85% homology to the sequence provided at NCBI Ref: NP_005521.1(human) or NP_083849.1 (murine). IDH3α may also be termed IDH3α. Anexemplary human amino acid sequence is provided below:

(SEQ ID NO: 41) MAGPAWISKVSRLLGAFHNPKQVIRGETGGVQTVTLIPGDGIGPEISAAVMKIFDAAKAPIQWEERNVTAIQGPGGKWMIPSEAKESMDKNKMGLKGPLKTPIAAGHPSMNLLLRKTFDLYANVRPCVSIEGYKTPYTDVNIVTIRENTEGEYSGIEHVIVDGVVQSIKLITEGASKRIAEFAFEYARNNHRSNVTAVHKANIMRMSDGLFLQKCREVAESCKDIKFNEMYLDTVCLNMVQDPSQFDVLVMPNLYGDILSDLCAGLIGGLGVTPSGNIGANGVAIFESVHGTAPDIAGKDMANPTALLLSAVMMLRHMGLFDHAARIEAACFATIKDGKSLTKDLGGNAK CSDFTEEICRRVKDLD

An exemplary murine amino acid sequence is provided below:

(SEQ ID NO: 42) MAGSAWVSKVSRLLGAFHNTKQVTRGFAGGVQTVTLIPGDGIGPEISASVMKIFDAAKAPIQWEERNVTAIQGPGGKWMIPPEAKESMDKNKMGLKGPLKTPIAAGHPSMNLLLRKTFDLYANVRPCVSIEGYKTPYTDVNIVTIRENTEGEYSGIEHVIVDGVVQSIKLITEEASKRIAEFAFEYARNNHRSNVTAVHKANIMRMSDGLFLQKCREVAENCKDIKFNEMYLDTVCLNMVQDPSQFDVLVMPNLYGDILSDLCAGLIGGLGVTPSGNIGANGVAIFESVHGTAPDIAGKDMANPTALLLSAVMMLRHMGLFDHAAKIEAACFATIKDGKSLTKDLGGNAK CSDFTEEICRRVKDLD

By “IDH3α polynucleotide” is meant a nucleic acid molecule encoding aIDH3α polypeptide. An exemplary human IDH3α polynucleotide sequence isprovided at NM_005530:

(SEQ ID NO: 43) 1 gttgctgcgg agccaggagg ggaagcgatg gctgggcccgcgtggatctc taaggtctct 61 cggctgctgg gggcattcca caacccaaaa caggtgaccagaggttttac tggtggtgtt 121 cagacagtaa ctttaattcc aggagatggt attggcccagaaatttcagc tgcagttatg 181 aagatttttg atgctgccaa agcacctatt cagtgggaggagcggaacgt cactgccatt 241 caaggacctg gaggaaagtg gatgatccct tcagaggctaaagagtccat ggataagaac 301 aagatgggct tgaaaggccc tttgaagacc ccaatagcagccggtcaccc atctatgaat 361 ttactgctgc gcaaaacatt tgacctttac gcgaatgtccgaccatgtgt ctctatcgaa 421 ggctataaaa ccccttacac cgatgtaaat attgtgaccattcgagagaa cacagaagga 481 gaatacagtg gaattgagca tgtgattgtt gatggagtcgtgcagagtat caagctcatc 541 accgaggggg cgagcaagcg cattgctgag tttgcctttgagtatgcccg gaacaaccac 601 cggagcaacg tcacggcggt gcacaaagcc aacatcatgcggatgtcaga tgggcttttt 661 ctacaaaaat gcagggaagt tgcagaaagc tgtaaagatattaaatttaa tgagatgtac 721 cttgatacag tatgtttgaa tatggtacaa gatccttcccaatttgatgt tcttgttatg 781 ccaaatttgt atggagacat ccttagtgac ttgtgtgcaggattgatcgg aggtctcggt 841 gtgacaccaa gtggcaacat tggagccaat ggggttgcaatttttgagtc ggttcatggg 901 acggctccag acattgcagg caaggacatg gcgaatcccacagccctcct gctcagtgcc 961 gtgatgatgc tgcgccacat gggacttttt gaccatgctgcaagaattga ggctgcgtgt 1021 tttgctacaa ttaaggacgg aaagagcttg acaaaagatttgggaggcaa tgcaaaatgc 1081 tcagacttca cagaggaaat ctgtcgccga gtaaaagatttagattaaca cttctacaac 1141 tggcatttac atcagtcact ctaaatggac accacatgaacctctgttta gaatacctac 1201 gtatgtatgc attggtttgc ttgtttcttg acagtacatttttagatctg gccttttctt 1261 aacaaaatct gtgcaaaaga tgcaggtgga tgtccctaggtctgttttca aagaactttt 1321 tccaagtgct tgttttattt attaagtgtc tacctggtaaatgttttttt tgtaaactct 1381 gagtggactg tatcatttgc tattctaaac cattttacacttaagttaaa atagtttctc 1441 ttcagctgta aataacagga tacagaatta acaagagaaaatgtctaact ttttaagaaa 1501 aaccttattt tcttcggttt ttgaaaaaca taatggaaataaaacaggat attgacataa 1561 tagcacaaaa tgacactctt ctaaaactaa atgggcacaagagaattttc ctgggaaagt 1621 tcacatcaaa aagagtgaat gtggtatatt tctaaatgatatggaaaata gagacagatt 1681 tgtcctttac agaaattact gagtgtgaat aaaaacttcagatccaagaa atatataatg 1741 agagatataa tttttgttaa taagacaaag gtaatatattggatacaaag acacaaatgt 1801 attgtgtgtt caattatttt gttgtcttga gatttaatattctttccaag agcttttaat 1861 gaagcagaga gctagtactt cattttcact ggatacattttcagcatcat gagttgtcac 1921 agcctctgag cccctgatct gaagccagaa gggctgagtgtattgtaaac ttattcttgc 1981 atgttgctgt ctgggaatgg accacactac agcaggtagttctgggggcg atactgccga 2041 aaggcccgaa cacatgtatt ttggctgcaa ttgaggaacttgggatgcta ttaattttgt 2101 atttcagcaa ctgccccttc tcctatccca aagcaccaattactgccctc tgcctcagca 2161 gtaccagtat aagatgacat tccaaagact ggaggcaactcagcctgagt taattcacaa 2221 aattatgcca tgctggggct tgagcttgag cttgggcttaggcttgggct cagcttttga 2281 ccctcaggca tctcctttcc cttcctgtct tcctctcccttctcctctgc tgcagcatga 2341 ttttcttaat cttcagacac tcactatttt catgaacagttaccctctgt ccccacaacc 2401 aaagacaact catggcctcc tttggccctt gtgtaacattgcaaacctgt ggctttgcaa 2461 aatgtaccca ggtcacaagg ggattttttt ttttttagcaatgatatccc tgtctgggtc 2521 actttttaag cttgtaaccg cccccccaga cttataatcttaaatgtatt ttcctttgtt 2581 taagctgctg cttcctctgt ttcattggat tgtgccagttatcagtggct cttgggttca 2641 aagtaataaa gaattccaaa actgaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 2701 a

An exemplary murine IDH3α polynucleotide sequence is provided atNM_029573:

(SEQ ID NO: 44) 1 gacgcgatgg ccgggtccgc gtgggtgtcc aaggtctctcggctgctggg tgcattccac 61 aacacaaaac aggtgacaag aggttttgct ggtggtgttcagacagtaac tttaattcct 121 ggagatggaa ttggcccaga aatttcagcc tcagtcatgaagatttttga tgctgccaaa 181 gcacctattc agtgggagga gcgcaatgtc acagcaattcaaggaccagg aggaaagtgg 241 atgatccctc cagaagccaa ggagtccatg gataagaacaagatgggctt gaaaggccca 301 ctaaagaccc caatagccgc tggccatcca tctatgaatctgttgcttcg taagacattt 361 gacctttatg ccaatgtccg gccatgtgtc tcaattgaaggttataaaac cccttacacg 421 gatgtaaata tcgtcaccat ccgagagaac acggaaggagaatacagtgg aattgagcat 481 gtgatcgttg atggggttgt gcagagcatc aagctcatcaccgaagaagc aagcaagcgc 541 attgcagagt ttgccttcga gtacgctcgg aacaaccaccggagcaacgt cacagctgtg 601 cacaaagcta acatcatgag gatgtcagat gggctctttctgcaaaaatg cagggaagtt 661 gcggagaact gtaaagacat taaatttaac gagatgtaccttgatactgt atgtttaaat 721 atggtacaag acccatccca gtttgatgtt cttgtcatgccaaatttata cggagacatc 781 cttagtgatc tgtgtgcagg actgattgga ggtcttggggtgactccaag tggcaatatt 841 ggagccaacg gtgttgccat ctttgaatcg gttcatggaacagccccgga cattgcaggc 901 aaggacatgg ccaaccccac ggccctcctg cttagtgctgtgatgatgct tcgccacatg 961 ggactttttg accatgcagc aaaaatcgag gctgcatgttttgctacaat taaggatgga 1021 aagagcttaa caaaagatct gggaggcaac gcgaagtgctctgacttcac agaagaaatc 1081 tgtcgtagag tcaaagactt agattagcac tcctgctggtggatttgctg cagtcagtca 1141 atcactccaa aaggataccc tgtaatcctc cttgagggcgcccaccattg gtttgcttgc 1201 ttcttgacag agtacgtttt ttgaatctgg ccttttcttaacaaaaccct tgcaatggat 1261 gcacatgatg gccccaggcc ttcattcaaa gggttttcccaagtgctggt tgtatttatt 1321 gtccgtctgg taaaccttat tttgtaaact gtaagtgaactgtatcattt atcattgtta 1381 acccatttta cacttcaggc aaaatcattt tcctcaactgtaaatattct gatacagaat 1441 taataagaga agatatttaa ctttttaaca aaagccctggatttttggtt tatgaaaaac 1501 aaactgggaa taaaacaggg ttttaacaat cgcacaagataacattattc taatactaat 1561 gggtacaaaa gaaatttact gggaaagttc acagcaaaaaaatggtatat ttcttaaaaa 1621 tatggaaata aagtatttgt cctatacatg aattactattaataaaaatg taagctccaa 1681 gaaatccata atgaatgatg taattttgtt actacatcggtaatccttgt caaggccccg 1741 gatgctctct gtgtatttga ttcttttgtt accttgagattcactatttt gggggaagag 1801 ctttcagata agggagatca ctcctcacta gacagatcgtcagcattgcg agctgtcagc 1861 catgagagcc agccactgca gatcccctcc cacgtggccacactccagcc agtgctgcag 1921 gtgaccctgg aaaggcctgg ctgccccttg actttccctaaagcaaccag tcactgcctt 1981 ctgccccagt agcacccatt acagacttaa ttgccgaggtggagctgact cagcccacgc 2041 tcatacaaat caggccaagc gggggcctgt gttaccagctgctgaccatc aggttctgcc 2101 cctcattctt cccacagcct ctgctccaca gcatgaacctagcctttggc ccacaccaaa 2161 gccaagctgt cttcccttag cccttgcact agtttgcaaactcgtggctt tgcataatgt 2221 accctggtcc caaggggatt tcttaacaac agatgtccctgtctgggtca tttttttaaa 2281 gcttttattt ggacttacaa tcttctgtgt attttactttaaaactgctg ctttccctgt 2341 ctcactggat tgttctggtt agcagtggct ttgggttcacagtaataaag aacttaagaa 2401 ctgaaaaaaa aaaaaaaa

By “IDH3β polypeptide” is meant a protein or fragment thereof having atleast 85% homology to the sequence provided at NCBI Ref: NP_008830.2(human) or NP_570954.1 (murine). IDH3β may also be termed IDH3b. Anexemplary human amino acid sequence is provided below:

(SEQ ID NO: 45) MAALSGVRWLTRALVSAGNPGAWRGLSTSAAAHAASRSQAEDVRVEGSFPVTMLPGDGVGPELMHAVKEVFKAAAVPVEFQEHHLSEVQNMASEEKLEQVLSSMKENKVAIIGKIHTPMEYKGELASYDMRLRRKLDLFANVVHVKSLPGYMTRHNNLDLVIIREQTEGEYSSLEHESARGVIECLKIVTRAKSQRIAKFAFDYATKKGRGKVTAVHKANIMKLGDGLFLQCCEEVAELYPKIKFETMIIDNCCMQLVQNPYQFDVLVMPNLYGNIIDNLAAGLVGGAGVVPGESYSAEYAVFETGARHPFAQAVGRNIANPTAMLLSASNMLRHLNLEYHSSMIADAVKKVIKVGKVRTRDMGGYSTTTDFIKSVIGHLQTKGS

An exemplary murine amino acid sequence is provided below:

(SEQ ID NO: 46) MAALSNVRWLTRAVLAARNSGAWRGLGTSTAHAASQSQAQDVRVEGAFPVTMLPGDGVGPELMHAVKEVFKAAAVPVEFKEHHLSEVQNMASEEKLEQVLSSMKENKVAIIGKIYIPMEYKGELASYDMQLRRKLDLFANVVHVKSLPGYKTRHNNLDLVIIREQTEGEYSSLEHESAKGVIECLKIVTRIKSQRIAKFAFDYATKKGRSKVTAVHKANIMKLGDGLFLQCCEEVAELYPKIKFETMIIDNCCMQLVQNPYQFDVLVMPNLYGNIIDNLAAGLVGGAGVVPGESYSAEYAVFETGARHPFAQAVGRNIANPTAMLLSATNMLRHLNLEYHSSMIADAVKKVIKAGKVRTRDMGGYSTTTDFIKSVIGHLHPHGG

By “IDH3β polynucleotide” is meant a nucleic acid molecule encoding aIDH3β polypeptide. An exemplary human IDH3β polynucleotide sequence isprovided at NM_006899:

(SEQ ID NO: 47) 1 gtcacttccc acgcgacttc ctgcgggaaa catggcggcattgagcggag tccgctggct 61 gacccgagcg ctggtctccg ccgggaaccc tggggcatggagaggtctga gtacctcggc 121 cgcggcgcac gctgcatcgc ggagccaggc cgaggacgtgagggtggagg gctcctttcc 181 cgtgaccatg cttccgggag acggtgtggg gcctgagctgatgcacgccg tcaaggaggt 241 gttcaaggct gccgctgtcc cagtggagtt ccaggagcaccacctgagtg aggtgcagaa 301 tatggcatct gaggagaagc tggagcaggt gctgagttccatgaaggaga acaaagtggc 361 catcattgga aagattcata ccccgatgga gtataagggggagctagcct cctatgatat 421 gcggctgagg cgtaagttgg acttatttgc caacgtagtccatgtgaagt cacttcctgg 481 gtatatgact cggcacaaca atctagacct ggtgatcattcgagagcaga cagaagggga 541 gtacagctct ctggaacatg agagtgcaag gggtgtgattgagtgtttga agattgtcac 601 acgagccaag tctcagcgga ttgcaaagtt cgcctttgactatgccacca agaaggggcg 661 gggcaaggtc actgctgtcc acaaggccaa catcatgaaacttggggatg ggttgttcct 721 gcagtgctgt gaggaagttg ctgaactgta ccccaaaatcaaatttgaga caatgatcat 781 agacaactgc tgcatgcagc tggtgcagaa tccttaccagtttgatgtgc ttgtgatgcc 841 caatctctat gggaacatta ttgacaatct ggctgctggcctggttgggg gagctggtgt 901 ggtccctggt gagagctata gtgcagaata cgcagtctttgagacgggtg cccggcaccc 961 atttgcccag gcagtgggca ggaatatagc caatcccacggccatgctgc tgtcggcttc 1021 caacatgctg cggcatctta atcttgagta tcactccagcatgatcgcag atgcggtgaa 1081 gaaggtgatc aaagttggca aggtgcggac tcgagacatgggcggctaca gcaccacaac 1141 cgacttcatc aagtctgtca tcggtcacct gcagactaaagggagctaga gccctttatt 1201 tcttccaacc ttgcaaggac cacactcccc atacccttcagtgcagtgta ccagggaaga 1261 gaccttgtgc ctctaagcag tggaccatgg tcaccttgctgggtagagcc taggttgtcc 1321 ttgggccggc ttccttaggg gacagactgt tgggtggtgatggggattgt taggatggag 1381 cccaggccac atggatgatg atgattctcc cccacaggttcgaacctctg acatgggtgg 1441 ctatgctact tgccatgact tcactgaggc tgtcattgctgccttgcccc acccataggc 1501 cctgtccata cccatgtaag gtgttcaata aagaacatgaaccaaaaaaa aaaaaaaaaa 1561 a

An exemplary murine IDH3β polynucleotide sequence is provided atNM_130884:

(SEQ ID NO: 48) 1 ggcgtcactt cccccgcgac ttcctcggcc gaacatggcagcgctgagca atgtccgctg 61 gctgacccga gcggtgctcg ccgctcggaa ctccggggcatggagaggtc tcggaacatc 121 tacggctcac gccgcttccc agagccaggc acaagatgtgagggtggagg gtgcctttcc 181 tgtgaccatg ctgcctggag acggcgtggg gccagagctcatgcatgctg tcaaggaagt 241 gttcaaggct gctgctgtcc ctgtggaatt taaggagcatcatctgagcg aggtgcagaa 301 tatggcttct gaggagaagc tggagcaggt gctgagttccatgaaggaga acaaagttgc 361 catcattgga aagatctata ccccaatgga gtataagggtgaactagcct cctatgatat 421 gcagctgagg cgtaagttgg atttgtttgc caacgtagtccacgtgaagt cacttcctgg 481 atacaagact cggcacaaca atctagacct ggttatcattcgagagcaga cagaagggga 541 gtatagctct ctggaacatg agagcgccaa gggtgtcattgagtgcctga agatcgtcac 601 tcgcaccaag tctcagagga ttgcaaagtt tgcgttcgactatgccacca agaaagggcg 661 gagcaaggtc acagccgtcc ataaagccaa catcatgaaactaggggatg gcttgttctt 721 gcagtgctgt gaggaagttg ctgaactgta ccctaaaatcaagtttgaaa ccatgatcat 781 agacaactgc tgcatgcagc tggtgcagaa cccttaccagtttgatgtgc tcgtgatgcc 841 caatctctat ggcaacataa ttgacaatct ggctgctggccttgttgggg gagctggcgt 901 ggttcctggg gagagctaca gtgcagagta tgcagtttttgagacgggtg ctcggcaccc 961 atttgcccag gcagtgggca ggaatatagc caaccccacagccatgctgc tgtcggccac 1021 caacatgctg cggcatctca atcttgagta tcactccagcatgattgcag atgcagtgaa 1081 gaaagtgatc aaagctggca aggtacggac tcgagacatgggaggctaca gcaccacaac 1141 tgacttcatc aagtctgtca tcggccacct gcacccccatgggggctaga gcccttactc 1201 cctccaattt caaaaggacc atgcttcgta tacatcccttcagtacaatg gaccagaaga 1261 gaacatctag acagtagact ataatagctt ttctgaggctaggctgtcct gggggctggt 1321 gttaagggta tctcaaaggg tgggttgttg cgacaaggcccagaccctaa gatgataact 1381 ttttcccaca ggttcgaacc tcagatatgg gtggttatgccacatgtcat gacttcactg 1441 aagctgtcat tactgccctg tcataaatcc tatacatgcccatgaaaaaa atagtcaata 1501 aacaaaatac acacatacta

By “IDH3γ polypeptide” is meant a protein or fragment thereof having atleast 85% homology to the sequence provided at NCBI Ref: NP_004126.1(human) or NP_032349.1 (murine). IDH3γ may also be termed IDH3g. Anexemplary human amino acid sequence is provided below:

(SEQ ID NO: 49) MALKVATVAGSAAKAVLGPALLCRPWEVLGAHEVPSRNIFSEQTIPPSAKYGGRHIVIMIPGDGIGPELMLHVKSVERHACVPVDFEEVHVSSNADEEDIRNAIMAIRRNRVALKGNIETNHNLPPSHKSRNNILRISLDLYANVIHCKSLPGVVIRHKDIDILIVRENTEGEYSSLEHESVAGVVESLKIITKAKSLRIAEYAFKLAQESGRKKVTAVHKANIMKLGDGLFLQCCREVAARYPQITFENMIVDNTTMQLVSRPQQFDVMVMPNLYGNIVNNVCAGLVGGPGLVAGANYGHVYAVFETATRNTGKSIANKNIANPTATLLASCMMLDHLKLHSYATSIRKAVLASMDNENMHTPDIGGQGTTSEAIQDVIRHIRVINGRAVEA

An exemplary murine amino acid sequence is provided below:

(SEQ ID NO: 50) MALKVAIAAGGAAKAMLKPTLLCRPWEVLAAHVAPRRSISSQQTIPPSAKYGGRHIVTMIPGDGIGPELMLHVKSVERHACVPVDFEEVHVSSNADEEDIRNAIMAIRRNRVALKGNIETNHNLPPSHKSRNNILRTSLDLYANVIHCKSLPGVVTRHKDIDILIVRENTEGEYSSLEHESVAGVVESLKIITKAKSLRIAEYAFKLAQESGRKKVTAVHKANIMKLGDGLFLQCCREVAAHYPQITFDSMIVDNTTMQLVSRPQQFDVMVMPNLYGNIVNNVCAGLVGGPGLVAGANYGHVYAVFETATRNIGKSIANKNIANPTATLLASCMMLDHLKLHSYATSIRKAVLASMDNENMHTPDIGGQGTISQAIQDIIRHIRIINGRAVEA

By “IDH3γ polynucleotide” is meant a nucleic acid molecule encoding aIDH3γ polypeptide. An exemplary human IDH3γ polynucleotide sequence isprovided at NM_004135:

(SEQ ID NO: 51) 1 ggggcccagc tggtcgcggt ccccccctca acatggcggcagcggtgctc taggcgccgg 61 aagggggcgt gaatcggtgc gaccgcgcgc gtgcgcagtaccgggtccgc gcctgtcccc 121 gaaacttcgc accccgtcga actctcgcga gagcggtatctgcgtgtcgg gacgtgcgga 181 ggctctcact ttccgtcatg gcgctgaagg tagcgaccgtcgccggcagc gccgcgaagg 241 cggtgctcgg gccagccctt ctctgccgtc cctgggaggttctaggcgcc cacgaggtcc 301 cctcgaggaa catcttttca gaacaaacaa ttcctccgtccgctaagtat ggcgggcggc 361 acacggtgac catgatccca ggggatggca tcgggccagagctcatgctg catgtcaagt 421 ccgtcttcag gcacgcatgt gtaccagtgg actttgaagaggtgcacgtg agttccaatg 481 ctgatgaaga ggacattcgc aatgccatca tggccatccgccggaaccgc gtggccctga 541 agggcaacat cgaaaccaac cataacctgc caccgtcgcacaaatctcga aacaacatcc 601 ttcgcaccag cctggacctc tatgccaacg tcatccactgtaagagcctt ccaggcgtgg 661 tgacccggca caaggacata gacatcctca ttgtccgggagaacacagag ggcgagtaca 721 gcagcctgga gcatgagagt gtggcgggag tggtggagagcctgaagatc atcaccaagg 781 ccaagtccct gcgcattgcc gagtatgcct tcaagctggcgcaggagagc gggcgcaaga 841 aagtgacggc cgtgcacaag gccaacatca tgaaactgggcgatgggctt ttcctccagt 901 gctgcaggga ggtggcagcc cgctaccctc agatcaccttcgagaacatg attgtggata 961 acaccaccat gcagctggtg tcccggcccc agcagtttgatgtcatggtg atgcccaatc 1021 tctatggcaa catcgtcaac aatgtctgcg cgggactggtcgggggccca ggccttgtgg 1081 ctggggccaa ctatggccat gtgtacgcgg tgtttgaaacagctacgagg aacaccggca 1141 agagtatcgc caataagaac atcgccaacc ccacggccaccctgctggcc agctgcatga 1201 tgctggacca cctcaagctg cactcctatg ccacctccatccgtaaggct gtcctggcat 1261 ccatggacaa tgagaatatg cacactccgg acatcgggggccagggcaca acatctgaag 1321 ccatccagga cgtcatccgc cacatccgcg tcatcaacggccgggccgtg gaggcctagg 1381 ctggccctag gaccttcttg gtttgctcct tggattccccttcccactcc agcaccccag 1441 ccagcctggt acgcagatcc cagaataaag caccttctccctagaaaaaa aaaaaaaaaa 1501 aa

An exemplary murine IDH3γ polynucleotide sequence is provided atNM_008323:

(SEQ ID NO: 52) 1 ggtgcttaat gttttgacct gtagaggtcc tcacttttcgtcatggcgct gaaggtggcg 61 atagctgctg gcggtgctgc aaaggcaatg ctcaagccaactctcctctg ccgtccttgg 121 gaggttctgg ctgcccatgt ggccccccga aggagcatttcctcacaaca aacaattcct 181 ccatctgcta agtatggtgg gcggcataca gtgactatgatcccagggga tggcatcggc 241 ccagagctca tgttgcatgt taagtctgta ttcaggcatgcatgtgtgcc ggtggacttt 301 gaagaggtgc atgtaagctc caacgctgat gaggaggacatccgcaatgc catcatggcc 361 atccgccgga accgtgtggc cctgaagggc aacattgaaacaaatcataa cctgccacca 421 tcccacaaat ctcgaaacaa catccttcgc accagcctagacctctatgc caacgtcatc 481 cactgtaaga gcctgccagg agtggtgacc cggcacaaggacatagacat cctcattgta 541 cgggaaaaca cagaaggcga gtacagcagc ctggagcatgagagcgtagc aggagtggtg 601 gagagcttga agattatcac caaagccaag tccctgcgcattgctgaata tgctttcaag 661 ctggcccagg agagtgggcg taagaaagtg acggctgtgcacaaggccaa catcatgaaa 721 ctgggtgatg gactcttcct ccagtgctgc agggaagtagcagcccacta ccctcagatc 781 acctttgaca gcatgattgt agacaacaca acaatgcagctggtatcccg gcctcagcag 841 tttgatgtca tggtgatgcc taatctctat ggtaacattgtcaacaacgt ctgtgcaggg 901 ctagttggag gcccaggcct tgtggctggg gccaactatggccatgtgta tgcagtattc 961 gagacagcta caaggaacac aggcaaaagt attgccaataagaacattgc taacccgact 1021 gccacactgc tagcaagctg catgatgcta gaccacctcaagctccactc ctatgccact 1081 tccatccgca aagctgtctt agcatccatg gacaatgaaaatatgcatac cccagatatt 1141 ggaggccagg gcaccacatc ccaagccatc caggacatcattcgtcatat ccgcatcatt 1201 aatggacggg ctgtggaggc ttagctatcc ctacagttttgctcagcttg tctgtaggac 1261 tctcttctca ctttagcact ccagctagct tgggggacaggacccagaat aaagccactt 1321 ctgttccaga aaaaa

By “IDH3 polynucleotide” is meant a nucleic acid molecule encoding aIDH3 polypeptide.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST®, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST® program may be used, with a probabilityscore between e-3 and e-100 indicating a closely related sequence.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, murine, ovine, orfeline.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1T (related to FIG. 2) are bar graphs, images and twoschematics showing that ERRs and PGC1α/β were direct targets ofreprogramming factors during early reprogramming. FIGS. 1A-1D depict bargraphs showing that mouse ERRα/γ and PGC1α/β were activated inretroviral reprogramming mouse embryonic fibroblasts (MEFs) at day 3,shown by qPCR results (n=3, *p<0.01, error bars show standard error ofthe mean (s.e.m.)). FIG. 1E is a bar graph showing that depleting ERRγin retroviral reprogramming MEFs after day 4 did not influencereprogramming efficiency (n=3, error bars show standard deviation(s.d).). FIG. 1F is a linear graph showing that reprogramming cells withERRα or ERRγ depletion by lentiviral shRNA showed a reducedproliferation rate. FIG. 1G shows two images of cell cultures of Nanogstaining of immortalized MEFs from wild-type (ERRγ+/+) or ERRγ knockout(ERRγ−/−) embryos after retroviral OSKM reprogramming. FIGS. 1H-1J arebar graphs showing that human ERRα and PGC1α/β were up-regulated inretroviral reprogramming human lung fibroblast IMR90 cells at day 5, butnot in adipose stem cells (ADSCs), IMR90, or pluripotent stem cells(n=3, *p<0.01, error bars show s.e.m.). FIGS. 1K-1M are bar graphs ofqPCR showing relative expression of ERRα, PGC-1α and PGC-1β in singlefactor infected cells (n=3, error bars show s.e.m.). FIG. 1N is aschematic representation of ERRα, PGC-1α and PGC-1β induction by Oct3/4,Sox2, Klf4 or c-Myc. FIG. 1O is a bar graph showing relativereprogramming efficiencies of doxycycline-inducible reprogramming MEFswith and without ERRγ over expression (Ad-ERRγ and Ad-GFP,respectively). Reprogramming efficiency based on alkaline phosphatasestaining at day 21 (n=6, error bars show s.d. **p<0.01). FIG. 1P is aschematic design of the lentiviral reporter which recapitulates thehuman ERRα enhancer activity. A 974 bp enhancer sequence (chr11:64072402-64073375) which covers the upstream and 5′UTR of the human ERRαgene was cloned into a lentiviral reporter which contains greenfluorescence protein (GFP) and luciferase. A separate constitutiveactive promoter EF1α drove the expression of Neomycin resistance gene,which allowed the selection in cells with low expression of endogenousERRα. FIG. 1Q is a schematic design of isolation of a sub-population ofreprogramming cells which has high ERRα expression. Human fibroblastswere transduced with lentiviral reprogramming factors which overexpressOct4, Sox2, Klf4, cMyc, Nanog and Lin28. The fibroblasts were transducedwith ERRα reporter at the same time. GFP was not observed at day 1-2,but started to appear and reach its peak around day 4-6. Cells weresorted by GFP intensity at this stage to isolate the top 5% GFP positivecells. FIG. 1R is a fluorescence image showing that the ERRα reportercould be observed in day 5 reprogramming fibroblast, whereas the controlwhich only transduced with reporter but not the reprogramming factorsremained GFP negative. FIG. 1S shows fluorescence activated cell sorting(FACS) results of reprogramming cells with ERRα reporter. P4 representthe GFP positive population. FIG. 1T shows gene expression comparingERRα and its targets in normal fibroblasts (control), fibroblaststransduced with reporter only (GF only), and GFP+ and GFP− population atreprogramming day 6. ERRα and its targets were highly enriched in GFP+population, compared to other samples, indicating that the ERRα reportercould fully capture the endogenous ERRα expression pattern.

FIGS. 2A-2J are bar graphs and images showing ERRα/γ and PGC1α/β wereimportant for induced pluripotency in both mouse and human cells. FIG.2A is a bar graph showing mouse embryonic fibroblasts (MEFs) undergoingretroviral reprogramming with OSKM were transduced with control, ERRα,ERRγ, PGC-1α or PGC-1β shRNA. Depletion of ERRα/γ and PGC-1α/βsignificantly reduced reprogramming efficiency. (n=3, error bars shows.d.). FIGS. 2B-2F depict images of cell cultures and graphs showingERRγlox/lox and ERRγlox/loxCreERT mouse MEFs infected with adoxycycline-inducible OSKM lentivirus that were treated with4-Hydroxytamoxifen (4-OHT) 3 days after OSKM induction. FIG. 2B-E arebright field images and graphs showing that ERRγ depletion reduced theclusters of early reprogramming cells (FIG. 2B), significantly reducedAP colonies (FIGS. 2C and 2D), and reduced Nanog-positive colonies(FIGS. 2E and 2F) (n=3, *p<0.01, error bars show s.d.). FIG. 2G is a bargraph showing that ERRα and PGC-1α/β were important for reprogramming ofIMR90 (n=3, *p<0.01, error bars show standard deviation (s.d)). FIGS. 2Hand 2I are bar graphs depicting qPCR results showing that depletion ofp53 lead to increased expression of human ERRα during reprogramming ofIMR90 cells (n=3, *p<0.01, error bars show s.e.m). FIG. 2J are twoimages of cell cultures showing Nanog staining of retroviralOSKM-infected MEFs with p53 (left), or p53 and ERRγ (right) shRNAvectors, demonstrating that loss of ERRγ resulted in complete collapseof reprogramming even with p53 depletion.

FIGS. 3A-3G are graphs and a heat map showing that ERRα/γ induced ametabolic transition in early reprogramming, which is important toinduced pluripotency. FIG. 3A is a graph showing that the time course ofoxygen consumption rate (OCR) and extracellular acidification rate(ECAR) in Dox-induced reprogramming mouse embryonic fibroblasts (MEFs),isolated from the single gene transgenic mouse, revealed that thereprogramming population experienced an early oxidative phosphorylation(OXPHOS) burst. FIG. 3B is a graph showing that mitostress test of earlyreprogramming MEFs in FIG. 3A showed increased basal OCR and maximalOXPHOS capacity.

FIG. 3C is a graph showing that relative gene expression of ERRα,coactivators PGC-1α and PGC-1β, and Nanog after retroviral OSKMinfection of IMR90 cells, measured by qPCR, indicated that theexpression pattern of ERRs and their cofactors coincide with themetabolic switch in early reprogramming (n=3, *p<0.01, error bars shows.e.m.). FIG. 3D is a heat map showing temporal expression of metabolicgenes during retroviral OSKM induced IMR90 reprogramming. FIG. 3E is agraph showing OCR and extracellular acidification rate (ECAR)measurements of control and ERRα knockdown retroviral reprogrammingIMR90 cells demonstrating that ERRα was important for the early OXPHOSburst in human cells. FIG. 3F. is a graph showing that OCR and ECARmeasurements of control and ERRγ knockdown retroviral reprogramming MEFcells demonstrated that ERRγ is important for the early OXPHOS burst inmouse cells. FIG. 3G is a graph showing that rotenone treatment, whichinhibits the OXPHOS burst, resulted in significant reduction ofretroviral reprogramming efficiency in IMR90, indicating that themetabolic switch was important. (n=3, *p<0.05, error bars show s.d.).

FIGS. 4A-4H (related to FIG. 3) are graphs and a heat map showingchanges in metabolic activity and proto-oncogene tyrosine-protein kinase(ROS) genes during reprogramming. FIG. 4A is a bar graph showingkinetics of maximal oxidative phosphorylation (OXPHOS) capacity indoxycycline-inducible reprogramming mouse embryonic fibroblasts (MEFs).Reprogramming cells at days 2 to 5 have higher OXPHOS capacity than MEFsand iPSCs. FIGS. 4B and 4C are linear graphs showing that time coursemeasurements of oxygen consumption rate (OCR, FIG. 4B) and extracellularacidification rate (ECAR, FIG. 4C) in retroviral reprogramming IMR90cells showed an up-regulated metabolic profile in early reprogramminghuman fibroblasts. FIGS. 4D-4F are bar graphs showing that in earlyretroviral reprogramming of IMR90 cells, NADH, ATP and NAD+/NADH levelswere changed (n=5, error bars show s.d. *p<0.01). FIG. 4G is a heat mapshowing that metabolic genes listed in FIG. 4D showed a similarexpression pattern between various human ES and iPS lines, in contrastto fibroblast (hFib) lines. FIG. 4H is a linear graph showing thedynamic expression pattern of ROS genes SOD2, NOX4 and CAT duringretroviral reprogramming of IMR90 cells (n=3, error bars show s.e.m.*p<0.01).

FIGS. 5A-5G are images, graphs and a table showing that ERRγ enrichedsub-population in early reprogramming represented bona fidereprogramming cells with significantly enhanced reprogrammingefficiency. FIG. 5A depicts two images showing Sca1 and CD34 labeledbona fide reprogramming cells. Retroviral OSKM-infected mouse embryonicfibroblasts (MEFs) stained for Sca1 (green) and CD34 (red) expression,and phase contrast image (right). Sca1−CD34− double negative (DN) cellswere demarcated by white dashed lines from phase contrast images. FIG.5B shows six representative phase contrast images of Sca1−CD34− cellsduring retroviral reprogramming. Arrowheads indicate a representative DNcolony. FIGS. 5C and 5D are bar graphs of qPCR demonstrating that ERRγand PGC-1β were enriched in the DN population (n=3, error bars shows.e.m. *p<0.01). FIGS. 5E and 5F are bar graphs showing thatfluorescence-activated cell sorting (FACS)-isolated DN populationexhibited higher extracellular acidification rate (ECAR, FIG. 5E) andoxygen consumption rate (OCR, FIG. 5F) than double positive (DP) orsingle positive (SP) population (n=4, *p<0.05, error bars show s.d.).FIG. 5G is a table showing that DN cells demonstrated significantlyhigher reprogramming efficiency (n=7, *p<0.05, **p<0.01).

FIGS. 6A-6H (related to FIG. 5) are graphs and images showingpluripotency assays and germline transmission of iPSCs from doublenegative (DN) population. FIG. 6A is a group of graphs showing flowcytometry analysis of Sca1 and CD34 expression in WT mouse embryonicfibroblasts (MEFs), retroviral OSKM-infected MEFs, iPSCs and embryonicstem cells (ESCs). FIG. 6B is a bar graph showing that Sca1− MEFs hadsimilar reprogramming efficiencies to Sca1+MEFs (n=6, error bars shows.d.). FIG. 6C shows an alkaline phosphatase staining and phase contrastimage of iPSCs from DN population. FIG. 6D shows three images ofimmunofluorescence of SSEA1 (PE), Nanog (FITC) and DNA (DAPI) in iPSCsoriginating from Sca1−CD34− cells. FIGS. 6E and 6F are bar graphsshowing q-PCR analysis of pluripotent marker genes (FIG. 6E) anddifferentiation marker genes (FIG. 6F) in undifferentiated anddifferentiated mouse ESCs and iPSCs. The scale for Cardiac a-actin andMtap2 corresponded with y-axis shaded in gray on the right. FIG. 6G isan image that shows an adult chimeric mouse obtained from an iPSC linederived from DN cell population sorted 5 days after OSKM infection. FIG.6H is an image that shows offspring of chimera crossed with a C56BL/6Nfemale (asterisk) showing pups with black coats (green arrows)originating from iPSC cells.

FIGS. 7A-7G depict a table, graph, heat maps and a schematic oftranscriptome analysis that revealed that ERRs orchestrated theup-regulation of a panel of oxidative phosphorylation (OXPHOS) relatedgenes and promoted the metabolic switch during early reprogramming.FIGS. 7A and 7B are a matrix and a graph showing RNA-Seq analysis thatrevealed that the genome-wide expression pattern of various cell typescould be grouped into pluripotent stem cells, mouse embryonicfibroblasts (MEFs) and intermediate retroviral reprogramming cells,demonstrated by distance matrix (FIG. 7A) and clustering analysis (FIG.7B). FIGS. 7C and 7D are heat maps showing the RNA-Seq patterns of asubset of key pluripotency markers (FIG. 7C) and cell cycle genes (FIG.7D) that revealed similarity between double negative (DN) cells andESCs, indicating that the DN population represented bona fide earlyreprogramming cells which were in the process of adopting inducedpluripotency. FIG. 7E is an expression heat map from RNA-Seq data thatshowed that DN cells had a unique pattern in metabolic genes thatrepresents a hyperenergetic state. FIG. 7F is a heat map of geneexpression from microarray in IMR90 cells after ERRα depletion, showingthat a significant portion of the OXPHOS program was directly influencedby ERRα in human fibroblast reprogramming. FIG. 7G is a schematicrepresentation of the role of ERRs and PGC1α/β in inducing the earlyOXPHOS burst and transition to induced pluripotency. The OXPHOS burstwas important for somatic cell reprogramming and transient activation ofERRs and their co-factors were epistatic to the roadblock ofp53/p21-induced senescence in reprogramming.

FIGS. 8A-8C (related to FIG. 7) are two pie charts and a table showingthat ERRα depletion affected oxidative phosphorylation (OXPHOS) burstduring reprogramming. FIGS. 8A and 8B are a pie chart and a table ofKEGG PATHWAY analysis, a process that maps molecular datasets, whichrevealed a panel of OXPHOS related genes in DN population at 5 daysafter infection, indicating up-regulation of ERRγ in bona fidereprogramming cells induced the transcription of OXPHOS program. Geneselection was based on a Bonferroni error threshold of αBonf=0.01. FIG.8C is a table of enrichment analysis on gene sets generated using GOANALYSIS, that shows that ERRα depletion in IMR90 cells inducedwidespread changes of genes involved in metabolic processes.

FIGS. 9A-9F depict a schematic, graphs, and an image that revealed thatERRs function through IDH and α-ketoglutarate to regulate reprogramming.FIG. 9A is a schematic to demonstrate the function of ERRs inreprograming. IDH3 gene encodes isocitrate dehydrogenase, whichcatalyzes the oxidation of isocitrate to α-ketoglutarate. H3K4Me2 standsfor H3 histone (H3) with its lysine at the fourth (4th) amino acidposition from the N-terminal of the protein (K4) dimethylated (Me2).H3K4Me3 stands for histone 3 with its lysine at the fourth (4th) aminoacid position from the N-terminal of the protein (K4) trimethylated(Me3). H3K4Me 1 stands for H3 histone with its lysine at the fourth(4th) amino acid position from the N-terminal of the protein (K4)monomethylated (Me1). H3K4 stands for H3 histone with its lysine at thefourth (4th) amino acid position from the N-terminal of the protein (K4)unmethylated.

FIG. 9B is a bar graph showing the NAD+/NADH ratio change duringreprogramming, corresponding with the surge of ERR expression. FIG. 9Cis a bar graph showing that IDH3 genes regulation in variousreprogramming populations. WT fibroblast stands for wild typefibroblast, which was not infected by lentivirus. Mock infection wasincluded as a control. ERRα−GFP describes a lentivirus encoding GFPprotein under the control of the ERRα promoter. Cells were eitheruntreated (WT fibroblasts), mock infected or infected with the ERRα−GFPlentivirus. ERRα−GFP infected cells were FACS stored based on GFPactivity (ERRα−GFP+ and ERRα−GFP−). The relative expression of IDH3genes in the various cell populations was determined by qPCR. FIG. 9D isa bar graph showing α-ketoglutarate level in early reprogramming (day 5)without (control) and with treatment with a small hairpin RNA (shRNA)designed to reduce the expression of ERRγ (ERRg shRNA). α-KG stands forα-ketoglutarate. FIG. 9E shows representative images of iPS coloniesafter treatment of D-2-hydroxyglutarate (D-2-HG) or L-2-hydroxyglutarate(L-2-HG). FIG. 9F is a bar graph showing that reprogramming efficiencyafter D-2-HG or L-2-HG treatment of the cells. The image and the barlabelled with “Veh” in FIGS. 9E and 9F represents the iPS colonies afternegative control treatment, in which the cells were treated with thesolvent for D-2-HG and L-2-HG only.

FIGS. 10A-10B depict a schematic and a table showing that ERRαexpression labels a metabolically active cell subpopulation during earlyreprogramming. FIG. 10A is schematic presentation of experimentaldesign. IMR90 cells are transduced with lentivirus expressingreprogramming factors Oct4, Sox2, Klf4, Myc, Lin28, and Nanog, togetherwith a lentiviral GFP reporter which reflect the endogenous ERRαactivity. Lenti-OSKMLN stands for lentivirus expressing Oct4, Sox2,Klf4, Myc, Lin28, and Nanog GF-hEERa-III stands for a lentiviral GFPreporter in which the GFP activity is a measure of the endogenous ERRαexpression pattern. Cells are sorted based on GFP expression in Day 2 toDay 6 and RNA sequencing was performed for the cells in allsub-populations. FIG. 10B is a table to show the results of KEGG geneontology analysis of the genes enriched in GFP+ population.

FIGS. 11A-11B are graphs showing the promoter/enhancer landscapes inERRα+ and ERRα− reprogramming populations. FIG. 11A are graphs showingthe H3K4Me2 level in the enhancer/promoter regions of of genes thatfunction in fibroblast identity, such as SNAI1 and ZEB2, in ERRα+ andERRα− population. FIG. 11B are graphs showing the H3K4Me2 level in theenhancer/promoter of genes that function in reprograming, such as Oct4and Sox2. H3K4Me2 stands for H3 histone with the lysine at the fourth(4th) position from the N-terminal of the protein which is dimethylated.

DETAILED DESCRIPTION OF THE INVENTION

As described below, the invention generally features compositionscomprising induced pluripotent stem cell progenitors (also termedreprogramming progenitor cells) and methods of isolating such cells. Theinvention also provides compositions comprising induced pluripotent stemcells (iPSCs) derived from such progenitor cells. Induced pluripotentstem cell progenitors generate iPSCs at high efficiency.

Cell metabolism is adaptive to extrinsic demands. However, the intrinsicmetabolic demands that drive the induced pluripotent stem cell (iPSC)program remain unclear. While glycolysis increases throughout thereprogramming process, here it was demonstrated that the estrogenrelated nuclear receptors (ERRα and γ) and their partnered co-factorsPGC-1α and β, were transiently induced at an early stage resulting in aburst of oxidative phosphorylation (OXPHOS) activity. Up-regulation ofERRα or γ was important for both the OXPHOS burst in human and mousecells, respectively, as well as in iPSC generation itself. Failure toinduce this metabolic switch collapsed the reprogramming process. Theinvention is based, at least in part, on the discovery of a rare pool ofSca1−/CD34− sortable cells that is highly enriched in bona fidereprogramming progenitors. Transcriptional profiling confirmed thatthese progenitors are ERRγ and PGC-1β positive and have undergoneextensive metabolic reprogramming. These studies characterize apreviously unrecognized, ERR-dependent metabolic gate prior toestablishment of induced pluripotency.

Accordingly, the invention provides compositions comprisingreprogramming progenitors or their descendants (i.e., IPSCs), andmethods of using such compositions for the treatment of conditionsassociated with a deficiency in cell number.

Induced Pluripotent Stem Cells

An understanding of the molecular mechanisms that influence thegeneration, maintenance, and differentiation of human pluripotent stemcells is key to advancing their use in a therapeutic setting. Whereasthe transcriptional and epigenetic dynamics have been extensivelydocumented, temporal changes in metabolic states during the induction ofpluripotency remain largely unknown. Distinct from somatic cells,pluripotent stem cells have unique metabolic pathways (Zhang et al.,2012, Cell stem cell 11, 589-595), which influence their cellularbehavior and epigenetic status. Indeed, factors involved in metabolicfunctions such as mitochondrial proteins are among the first to beup-regulated in cells undergoing reprogramming. Therefore, delineatingthe molecular mechanisms governing the dynamic regulation of cellularmetabolism is crucial to understanding the connections between metabolicand epigenetic reprogramming.

Nuclear receptors (NRs) are pleiotropic regulators of organ physiologycontrolling broad aspects of glucose and fatty acid metabolism andoverall energy homeostasis (Mangelsdorf et al., 1995, Cell 83, 835-839,Yang et al., 2006, Cell 126, 801-810). While orphan receptors such asthe Estrogen Related Receptors (ERRs) are ligand-independent, theynonetheless are capable of directing dramatic changes in both glycolyticand oxidative metabolism in tissues with high energy. ERRs switchbetween various oxidative states by associating preferentially withtheir co-activators PGC-1α/β. The ERR family member ERRβ (also known asEsrrb) is glycolytic in the absence of PGC-1α and plays a key role inestablishing pluripotency (Buganim et al., 2012, Cell 150, 1209-1222;Feng et al., 2009, Nature cell biology 11, 197-203; Festuccia et al.,2012, Cell stem cell 11, 477-490; Martello et al., 2012, Cell stem cell11, 491-504). In contrast, ERRα and ERRγ, which are expressed inoxidative tissues such as skeletal muscle and heart (Narkar et al.,2011, Cell Metab 13, 283-293), have not previously been linked to iPSCgeneration. As described in detail below, transient up-regulation ofERRα and γ in the early stages of reprogramming induced a uniqueenergetic state. Furthermore, it was shown that the transient OXPHOSburst and increased glycolysis initiated by this metabolic switch wereimportant for epigenetic reprogramming. Mechanistically, ERRα and γ wereenriched in bona fide reprogramming progenitors and induced widespreadchanges in metabolic gene networks. These results indicate that anERR-mediated metabolic transition is important for induced pluripotency.

Accordingly, the invention provides methods for generating areprogramming progenitor that is capable of giving rise to inducedpluripotent stem cells at high efficiency. In one embodiment, aSca1−CD34− reprogramming progenitor is approximately 50-fold moreefficient at generating iPSCs than a reference cell. In otherembodiments, nearly 75% of the iPSC colonies in a population weregenerated by Sca1−CD34− reprogramming progenitors which were less than5% of the OSKM infected cells. Surprisingly, Sca1−CD34− reprogrammingprogenitors exhibited a 1500% increased colony formation frequency (CFF)relative to a reference cell.

Cellular Compositions

Compositions of the invention comprising purified reprogrammingprogenitors or induced pluripotent stem cells derived from thoseprogenitors can be conveniently provided as sterile liquid preparations,e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions,or viscous compositions, which may be buffered to a selected pH. Liquidpreparations are normally easier to prepare than gels, other viscouscompositions, and solid compositions. Additionally, liquid compositionsare somewhat more convenient to administer, especially by injection.Viscous compositions, on the other hand, can be formulated within theappropriate viscosity range to provide longer contact periods withspecific tissues. Liquid or viscous compositions can comprise carriers,which can be a solvent or dispersing medium containing, for example,water, saline, phosphate buffered saline, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like) and suitablemixtures thereof.

Sterile injectable solutions can be prepared by incorporating thereprogramming progenitors or their progeny utilized in practicing thepresent invention in the required amount of the appropriate solvent withvarious amounts of the other ingredients, as desired. Such compositionsmay be in admixture with a suitable carrier, diluent, or excipient suchas sterile water, physiological saline, glucose, dextrose, or the like.The compositions can also be lyophilized. The compositions can containauxiliary substances such as wetting, dispersing, or emulsifying agents(e.g., methylcellulose), pH buffering agents, gelling or viscosityenhancing additives, preservatives, flavoring agents, colors, and thelike, depending upon the route of administration and the preparationdesired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”,17th edition, 1985, incorporated herein by reference, may be consultedto prepare suitable preparations, without undue experimentation.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin. According to the present invention,however, any vehicle, diluent, or additive used would have to becompatible with the reprogramming progenitors or their descendants.

The compositions can be isotonic, i.e., they can have the same osmoticpressure as blood and lacrimal fluid. The desired isotonicity of thecompositions of this invention may be accomplished using sodiumchloride, or other pharmaceutically acceptable agents such as dextrose,boric acid, sodium tartrate, propylene glycol or other inorganic ororganic solutes. Sodium chloride is preferred particularly for bufferscontaining sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the agent selected. Theimportant point is to use an amount that will achieve the selectedviscosity. Obviously, the choice of suitable carriers and otheradditives will depend on the exact route of administration and thenature of the particular dosage form, e.g., liquid dosage form (e.g.,whether the composition is to be formulated into a solution, asuspension, gel or another liquid form, such as a time release form orliquid-filled form).

Those skilled in the art will recognize that the components of thecompositions should be selected to be chemically inert and will notaffect the viability or efficacy of the reprogramming progenitors ortheir descendants (i.e., IPSCs) as described in the present invention.This will present no problem to those skilled in chemical andpharmaceutical principles, or problems can be readily avoided byreference to standard texts or by simple experiments (not involvingundue experimentation), from this disclosure and the documents citedherein.

One consideration concerning the therapeutic use of reprogrammingprogenitors or their descendants (i.e., IPSCs) of the invention is thequantity of cells necessary to achieve an optimal effect. The quantityof cells to be administered will vary for the subject being treated. Inone embodiment, between 10⁴ to 10⁸, between 10⁵ to 10⁷, or between 10⁶and 10⁷ cells of the invention are administered to a human subject. Inpreferred embodiments, at least about 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, and5×10⁷ cells of the invention are administered to a human subject. Theprecise determination of what would be considered an effective dose maybe based on factors individual to each subject, including their size,age, sex, weight, and condition of the particular subject. Dosages canbe readily ascertained by those skilled in the art from this disclosureand the knowledge in the art.

The skilled artisan can readily determine the amount of cells andoptional additives, vehicles, and/or carrier in compositions and to beadministered in methods of the invention. Typically, any additives (inaddition to the active stem cell(s) and/or agent(s)) are present in anamount of 0.001 to 50% (weight) solution in phosphate buffered saline,and the active ingredient is present in the order of micrograms tomilligrams, such as about 0.0001 to about 5 wt %, preferably about0.0001 to about 1 wt %, still more preferably about 0.0001 to about 0.05wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10wt %, and still more preferably about 0.05 to about 5 wt %. Of course,for any composition to be administered to an animal or human, and forany particular method of administration, it is preferred to determinetherefore: toxicity, such as by determining the lethal dose (LD) andLD50 in a suitable animal model e.g., rodent such as mouse; and, thedosage of the composition(s), concentration of components therein andtiming of administering the composition(s), which elicit a suitableresponse. Such determinations do not require undue experimentation fromthe knowledge of the skilled artisan, this disclosure and the documentscited herein. And, the time for sequential administrations can beascertained without undue experimentation.

Administration of Cellular Compositions

Compositions comprising reprogramming progenitors or their descendants(i.e., IPSCs) are described herein. In particular, the inventionprovides for the administration of an induced pluripotent stem cellderived from a reprogramming progenitor that expresses ERRalpha or gammaand optionally PGC1 alpha or beta. Such cells can be providedsystemically or locally to a subject for the treatment or prevention ofa disease or condition associated with a decrease in cell number (e.g.,neurodegenerative diseases, heart disease, autoimmune diseases, type Idiabetes, type II diabetes, pre-diabetes, metabolic disorders, and thetreatment of other diseases or disorders associated with a deficiency incell division, differentiation and cell death (e.g., a reduction in thenumber of pancreatic cells, a reduction of T-cells, a loss of neuronalcells or myocytes). In one embodiment, cells of the invention aredirectly injected into an organ or tissue of interest (e.g., pancreas,thymus, brain, muscle, or heart). Alternatively, compositions comprisingcells of the invention are provided indirectly to the organ of interest,for example, by administration into the circulatory system (e.g., thecardio or pancreatic vasculature). Expansion and differentiation agentscan be provided prior to, during or after administration of the cells toincrease production of cells having, for example neurotransmitter, orinsulin producing potential in vitro or in vivo. The cells can beadministered in any physiologically acceptable vehicle, normallyintravascularly, although they may also be introduced into anotherconvenient site where the cells may find an appropriate site forregeneration and differentiation.

In one approach, at least 100,000, 250,000, or 500,000 cells areinjected. In other embodiments, 750,000, or 1,000,000 cells areinjected. In other embodiments, at least about 1×10⁵ cells will beadministered, 1×10⁶, 1×10⁷, or even as many as 1×10⁸ to 1×10¹⁰, or moreare administered. Selected cells of the invention comprise a purifiedpopulation of cells that express ERRalpha or gamma and PGC1 alpha orbeta. Preferable ranges of purity in populations comprising selectedcells are about 50 to about 55%, about 55 to about 60%, and about 65 toabout 70%. More preferably the purity is at least about 70%, 75%, or 80%pure, more preferably at least about 85%, 90%, or 95% pure. In someembodiments, the population is at least about 95% to about 100% selectedcells. Dosages can be readily adjusted by those skilled in the art(e.g., a decrease in purity may require an increase in dosage). Thecells can be introduced by injection, catheter, or the like.

Compositions of the invention include pharmaceutical compositionscomprising reprogramming progenitors or their descendants (i.e., IPSCs)and a pharmaceutically acceptable carrier. Administration can beautologous or heterologous. For example, somatic cells can be obtainedfrom one subject, and administered to the same subject or a different,compatible subject.

Selected cells of the invention or their progeny (e.g., in vivo, ex vivoor in vitro derived) can be administered via localized injection,including catheter administration, systemic injection, localizedinjection, intravenous injection, or parenteral administration. Whenadministering a therapeutic composition of the present invention (e.g.,a pharmaceutical composition containing a selected cell), it willgenerally be formulated in a unit dosage injectable form (solution,suspension, emulsion).

Accordingly, the invention also relates to a method of treating asubject having, for example, a disease or condition characterized by adeficiency in cell number, including but not limited toneurodegenerative diseases, cancer, heart disease, autoimmune diseases,type I diabetes, type II diabetes, pre-diabetes, metabolic disorders,and the treatment of other diseases or disorders associated with adeficiency in cell division, differentiation and cell death (e.g., areduction in the number of pancreatic cells, a reduction of T-cells, aloss of neuronal cells or myocytes). This method comprises administeringto the subject an effective amount either of a reprogramming progenitoror descendant thereof (i.e., IPSCs) isolated as explained herein.

Kits

The invention provides kits comprising an effective amount ofreprogramming progenitors or their descendants (i.e., IPSCs). In oneembodiment, the invention provides a reprogramming progenitor derivedfrom an embryonic fibroblasts (MEFs) or a lung fibroblast that expressesERRalpha or gamma. Optionally, the cells also express PGC1α or β. Thecells are provided in unit dosage form. In some embodiments, the kitcomprises a sterile container which contains a therapeutic orprophylactic cellular composition; such containers can be boxes,ampules, bottles, vials, tubes, bags, pouches, blister-packs, or othersuitable container forms known in the art. Such containers can be madeof plastic, glass, laminated paper, metal foil, or other materialssuitable for holding medicaments.

If desired a cell of the invention is provided together withinstructions for administering the cell to a subject having or at riskof developing a condition characterized by a deficiency in cell number,such as a neurodegenerative disease, heart disease, autoimmune disease,type I diabetes, type II diabetes, pre-diabetes, other metabolicdisorders, or other diseases or disorders associated with a deficiencyin cell division, differentiation and cell death (e.g., a reduction inthe number of pancreatic cells, a reduction of T-cells, a loss ofneuronal cells or myocytes). The instructions will generally includeinformation about the use of the composition for the treatment orprevention of a neurodegenerative disease, cancer, heart disease,autoimmune disease, type I diabetes, type II diabetes, pre-diabetes,other metabolic disorders, or other diseases or disorders associatedwith a deficiency in cell division, differentiation and cell death(e.g., a reduction in the number of pancreatic cells, a reduction ofT-cells, a loss of neuronal cells or myocytes). In other embodiments,the instructions include at least one of the following: description ofthe cells; dosage schedule and administration for treatment orprevention of a neurodegenerative disease, cancer, heart disease,autoimmune disease, type I diabetes, type II diabetes, pre-diabetes,other metabolic disorders, or other diseases or disorders associatedwith a deficiency in cell division, differentiation and cell death orsymptoms thereof; precautions; warnings; indications;counter-indications; overdosage information; adverse reactions; animalpharmacology; clinical studies; and/or references. The instructions maybe printed directly on the container (when present), or as a labelapplied to the container, or as a separate sheet, pamphlet, card, orfolder supplied in or with the container.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1: ERRα/γ are Important for Somatic Cell Reprogramming

Temporal gene expression studies in mouse embryonic fibroblasts (MEFs)after reprogramming with Oct4, Sox2, Klf4 and cMyc (OSKM) or OSKrevealed transient increases in the expression of ERRγ, PGC-1α, PGC-1β,and to a lesser extent, ERRα, 3 days after infection (FIGS. 1A-1D).Furthermore, depletion of ERRγ, PGC-1α or PGC-1β by shRNA knockdowncoincident with OSKM induction significantly reduced reprogrammingefficiency in MEFs (FIG. 2A), whereas ERRγ depletion later inreprogramming had little effect (FIG. 1E). To further explore the timingof gene induction in early reprogramming, OSKM expression was induced inMEFs isolated from ERRγlox/lox and ERRγlox/lox CreERT mice viadoxycycline-inducible lentiviruses (Wei et al., 2009, Stem cells(Dayton, Ohio) 27, 2969-2978). While tamoxifen-treated ERRγlox/lox MEFs(ERRγ control cells) exhibited multiple foci of reprogramming cells 5days after doxycycline-induced OSKM expression, ERRγlox/lox CreERT MEFstreated with tamoxifen at day 3 (ERRγ iKO cells) displayedfibroblast-like morphology (FIG. 2B). Consistent with a failure of theERRγ iKO cells to reprogram, few alkaline phosphatase (AP) orNanog-positive colonies were observed after 3 weeks of OSKM infection,whereas control cells showed normal reprogramming efficiency (FIGS.2C-2F). As depletion of ERRγ or ERRα in reprogramming cells lead to areduction in cell proliferation (FIG. 1F), the reprogrammingefficiencies of immortalized MEFs generated from ERRγ knockout (ERRγ−/−)or wildtype (ERRγ+/+) mouse embryos were also compared. NoNanog-positive cells were detected in (ERRγ−/−) cells after OSKMinfection (FIG. 1G). Reprogramming efficiencies of doxycycline-induciblereprogramming MEFs with and without ERRγ over expression (Ad-ERRγ andAd-GFP, respectively) were also compared indicating that ERRγ overexpression significantly increased reprogramming efficiency (FIG. 1O)Together, these findings indicate that the induction of ERRγ early inreprogramming was important for iPSC generation from efficiency in MEFs.

Similar gene expression patterns were observed during the reprogrammingof human lung fibroblast IMR90 cells and adipose-derived stem cells(ADSCs), with the distinction that ERRα, rather than ERRγ, wasup-regulated (FIGS. 1H-1J). Parallel shRNA knockdown studies in thehuman IMR90 cells revealed a strong dependence on ERRα expression,alongside PGC-1α and β expression, whereas depletion of ERRγ waspartially tolerated (˜40% reduction in Nanog+ colonies, FIG. 2G),further indicating that ERRα rather than ERRγ was important for iPSCgeneration in human fibroblasts. Furthermore, knockdown of p53,previously shown to increase iPSC generation (Kawamura et al., 2009,Nature 460, 1140-1144), resulted in the hyper-induction of ERRα andNanog during IMR90 cell reprogramming (FIGS. 2H and 2I). Notably, thecoincident knockdown of ERRγ and p53 blocked iPSC generation in MEFs(FIG. 2J), indicating that the ERR signaling pathway was epistatic top53-induced senescence in iPSC reprogramming.

To decipher the molecular mechanisms driving ERR/PGC-1 induction, IMR90cells were infected with each of the four factors individually.Distinctive expression patterns for ERRα, PGC-1α and -1β were observed 5days after infection. Klf4, c-Myc and Sox2 were each able to efficientlyinduce ERRα, Oct3/4 and Klf4 both induced the expression of PGC-1α,while c-Myc efficiently induced PGC-1β expression (FIGS. 1K-1M). Thesepatterns of gene induction indicate that all four reprogramming factorscontributed in complementary ways to produce the operational ERRαtranscriptional complex at day 5 (FIG. 1N).

Further, the human ERRα gene was cloned into a lentiviral reporter whichcontained green fluorescence protein (GFP) and luciferase (FIG. 1P). Aseparate constitutive active promoter EF1α drove the expression ofNeomycin resistance gene, which allowed the selection in cells with lowexpression of endogenous ERRα (FIG. 1P). A sub-population ofreprogramming cells which had high ERRα expression were isolated (FIG.1Q). Human fibroblasts were transduced with lentiviral reprogrammingfactors which overexpressed Oct4, Sox2, Klf4, cMyc, Nanog and Lin28(FIG. 1Q). The fibroblasts were transduced with ERRα reporter at thesame time. GFP was not observed at day 1-2, but started to appear andreach its peak around day 4-6 (FIG. 1Q). Cells were sorted by GFPintensity at that stage to isolate the top 5% GFP positive cells (FIG.1Q). ERRα reporter could be observed in day 5 reprogramming fibroblast,whereas the control which only transduced with reporter but not thereprogramming factors remained GFP negative (FIG. 1R). Reprogrammingcells with ERRα reporter were analyzed by fluorescence activated cellsorting (FACS), P4 representing the GFP positive population (FIG. 1S).Gene expression between ERRα and its targets in normal fibroblasts(control), fibroblasts transduced with reporter only (GF only), and GFP+and GFP− population at reprogramming day 6 was compared (FIG. 1T). ERRαand its targets were highly enriched in GFP+ population, compared toother samples, indicating that the ERRα reporter could fully capture theendogenous ERRα expression pattern (FIG. 1T).

Example 2: ERRs Directed a Transient Hyper-Energetic State thatFunctions in Reprogramming

The increased expression of ERRs and their co-activators led to thequestion of whether acutely altered energy flux in the mitochondria maybe fueling reprogramming. Mouse embryonic fibroblasts (MEFs) from thereprogramming factor doxycycline-inducible mouse (Carey et al., 2010,Nature methods 7, 56-59) reached an oxidative phosphorylation (OXPHOS)peak around days 2-4 after induction (FIG. 3A). Importantly, the maximalOXPHOS capacity was also significantly increased in early reprogrammingMEFs (FIGS. 3B and 4A). A similar bioenergetics time course recorded ondays 3 to 10 after OSKM infection in human IMR90 cells revealed atransient increase in mitochondrial OXPHOS that peaked 5 days afterinfection (2.5-5.0 fold increase in oxygen consumption rates (OCR))accompanied by a sustained increase in glycolysis (2.5-3.5 fold increasein the extra-cellular acidification rates (ECAR)) (FIGS. 4B and 4C).Corresponding with the increased expression of energy regulators, thelevels of both nicotinamide adenine dinucleotide (NADH) and cellular ATPwere increased in IMR90 cells 5 days after infection, while theNAD+/NADH ratio decreased (FIGS. 4D-4F). Together, these resultsindicated that early reprogramming cells were in a hyper-energeticstate. Closer examination of human lung fibroblast IMR90 cells revealedremarkably coincident temporal expression patterns of ERRα, PGC-1α and βduring the early stages of reprogramming that are consistent with theknown role of PCG1α/β as an ERR cofactor (days 3 to 8, FIG. 3C). ERRsand PGC-1s directly regulate an extensive network of genes controllingenergy homeostasis including proteins involved in fatty acid oxidation,the tricarboxylic acid (TCA) cycle and OXPHOS. Therefore, the temporalexpression pattern of various known regulators of cellular energyhomeostasis during the reprogramming of IMR90 cells was examined.Remarkably, multiple key players in energy metabolism, including ATPsynthase in mitochondria (ATP5G1), succinate dehydrogenase (SDHB),isocitrate dehydrogenase (IDH3A) and NADH dehydrogenase (NDUFA2),reached peak expression at day 5 (FIGS. 3D and 4G). In addition, theinduction of superoxide dismutase 2 (SOD2), NADPH oxidase 4 (NOX4) andcatalase (CAT) by OSKM infection (FIG. 4H), indicated that theantioxidant program was being triggered coordinately with the ERRα−PGC-1surge.

Pluripotent stem cells are known to mainly rely on glycolysis to produceenergy. Previous studies have focused on the changes in glycolyticactivity during reprogramming, as elevated glycolysis was linked to afaster cell cycle and iPSC generation (Folmes et al., 2011, Cellmetabolism 14, 264-271; Panopoulos et al., 2012, Cell research 22,168-177; Shyh-Chang et al., 2013b, Science, New York, N.Y., 339,222-226). However, the present findings indicate that iPSC precursorsunderwent a transient increase in oxidative phosphorylation activity.The dynamics of ECAR support previous work showing that the glycolyticactivity of the cells was gradually enhanced and maintained duringreprogramming to a level similar to iPSCs (FIGS. 3A and 4C). Incontrast, the transient burst of OXPHOS during reprogramming of bothhuman and mouse cells had not been previously documented (FIGS. 3A, 3Band 4B). This led to the investigation of the potential influence of theERRα/γ surge on cell plasticity during reprogramming.

To examine a potential causal relationship between ERR expression andthe induction of the hyper-energetic state, the metabolic activities ofpartially reprogrammed cells before and after targeted shRNA knockdownswere compared. Notably, the increase in OXPHOS and glycolysis wascompletely abrogated in cells depleted of ERRs (ERRα in IMR90 cells atday 5, and ERRγ in MEFs at day 3; FIGS. 3E and 3F). Furthermore, themitochondrial inhibitor Rotenone significantly reduced iPSC generation,though only when treatment was coincident with the observedhyper-energetic state, consistent with the OXPHOS burst being necessaryfor reprogramming (FIG. 3G). Together these data indicate that ERRα andγ regulate iPSC generation through the induction of a transient enhancedmetabolic state that is important for somatic cell reprogramming.

Example 3: Bona Fide iPSC Progenitors were Enriched for ERRγ Expression

Under standard conditions, only a small percentage of cells aresuccessfully reprogrammed into iPSCs. Given the observation of ametabolic switch in the heterogeneous cell populations present in theearly stages of reprogramming, it was hypothesized that thesub-population of bona fide iPSC progenitors might be enriched for theERR-mediated hyper-energetic burst. Analysis of cell surface markersdifferentially expressed during mouse embryonic fibroblasts (MEFs)reprogramming revealed that early clusters of reprogramming cells lackedthe expression of stem cell antigen 1 (Sca1) and cluster ofdifferentiation gene 34 (CD34) expression (FIGS. 5A and 5B). Upon OSKMinduction, CD34 expression was promptly up-regulated, resulting in threedistinct cell sub-populations in early reprogramming cells; Sca1−CD34−double negative (DN), Sca1+CD34+ double positive (DP), and Sca1+CD34−single positive (SP) (FIG. 6A). Correlating with immunofluorescencestaining (FIG. 5A), only a minor fraction (˜3-5%) of early reprogrammingcells were Sca1−CD34− (FIG. 6A). Strikingly, ERRγ and PGC-1β expressionwere ˜10− and ˜7-fold higher, respectively, in the early reprogrammingDN cells compared to DP or SP cells, as determined by qPCR analysis(FIGS. 5C and 5D). Importantly, these early reprogramming DN cellsexhibited significantly elevated extracellular acidification rate (ECAR)and oxygen consumption rate (OCR) compared to DP or SP populations(FIGS. 5E and 5F), consistent with Sca1−CD34− labeling a subpopulationof hyper-energetic cells. Notably, Sca1−CD34− cells present innon-infected MEFs did not show elevated reprogramming efficiency (FIG.6B). To test the hypothesis that this hyper-energetic state is importantfor reprogramming, the number of iPS colonies generated from isolatedDN, SP and DP cells was compared. While DN cells comprised only ˜5% ofthe infected cells, they were approximately 50-fold more efficient atgenerating iPSCs than the DP or SP populations, based on Nanog staining(FIG. 5G; 35.5% (DN) vs 0.6% (DP) or 0.8% (SP)). That is, nearly 75% ofthe iPSC colonies generated were derived from less than 5% of theinfected cells, corresponding to a 1500% increased colony formationfrequency (CFF). The iPSCs derived from the DN population showedESC-like morphology and expressed high levels of alkaline phosphataseactivity as well as pluripotency markers (FIGS. 6C-6E). In addition,embryoid body differentiation of the DN-derived iPSCs produced markersfrom each of the three germ layers (FIG. 6F). Moreover, iPSCs generatedfrom DN cells contributed to the formation of chimeric mice withsubsequent crosses demonstrating germline-competency (FIGS. 6G and 6H).Collectively, these data indicate that the hyper-energetic cellsidentified in early reprogramming represented by the DN population, werebona fide reprogramming precursors that generate iPSCs at highefficiency.

Example 4: Reprogramming Cells Underwent an ERR-Mediated OXPHOS Burst

To better understand the molecular underpinnings of cell reprogrammingand cell fate determination, the complete transcriptomes, determined byRNA-Sequencing, of somatic fibroblasts (non-infected mouse embryonicfibroblasts (MEFs), mock infected MEFs at day 5), intermediatereprogramming cell populations (DN, DP, SP, unsorted day 5 cells) andpluripotent stem cells (iPSCs generated from the DN population andmESCs) were compared. Not unexpectedly, distance matrix and clusteringanalyses grouped the cell types into the above 3 categories (FIGS. 7Aand 7B). The clear separation of the DN population from the pluripotentstem cells indicated that these transitional cells have yet to adopt adurable pluripotency fate. Furthermore, the more subtle separation ofthe DN population from the other intermediate reprogramming cells in thecluster analysis indicated that they should express a unique genesignature associated with enhanced reprogramming efficiency (FIG. 7B).Indeed, the expression of selected pluripotency markers and key cellcycle genes in the DN population more closely resembled that observed inESCs and iPSCs than found in the DP and SP populations (FIGS. 7C and7D). However, a majority of other stem cell markers including ERRβ andNanog were not enriched in the DN population. Thus, the DN cellpopulation is in a definable transcriptional and metabolic state thatappeared to facilitate efficient progression toward pluripotency.

Pivotal pathways controlling the enhanced reprogramming efficiency of DNcells were identified by comparing transcriptomes between DN, DP or SPpopulations. Interestingly, KEGG PATHWAY analysis, a process that mapsmolecular datasets, of the differentially regulated genes identified(oxidative phosphorylation) OXPHOS as the most significantly alteredpathway in DN cells (FIGS. 8A and 8B). Furthermore, a comparison of theexpression levels of genes involved in cellular energy metabolismrevealed that the majority were upregulated in the DN population (FIG.7E), consistent with the DN population comprising the mosthyper-energetic cells. This supported the idea that a key feature ofbona fide reprogramming is directing progenitors to enter ahyper-energetic state.

Finally, to determine if a causal association exists between the ERRsurge and the increased expression of energy metabolism genes, thetranscriptional consequences of ERRα knockdown in reprogramming IMR90swere examined. The expression of a large number (1061) of metabolicgenes was significantly affected by ERRα depletion (FIG. 8C). Inparticular, dramatic decreases in the expression of regulators ofcellular energy homeostasis including NADH dehydrogenases (NDUF),succinate dehydrogenases (SDH), mitochondrial respiratory chains (COX),ATPase, and ATP synthases in mitochondria were seen (FIG. 7F). The factthat ERRα depletion influenced the expression of a plethora ofmitochondrial genes, including a variety of genes in Complex I-V, andthe TCA cycle (FIG. 7F), further supported the conclusion that transientERRα/γ expression induced an equally transient OXPHOS burst,facilitating reprogramming and enabling the transition from the somaticto pluripotent state (FIG. 7G).

Recent single-cell expression analyses revealed a requirement for earlyexpression of ERRβ (Buganim et al., 2012), previously demonstrated byFeng et al. to be a ‘Myc substitute’ (Feng et al., 2009). In this model,Sox2 and ERRβ mutually enhanced each other's expression and initiatedthe reprogramming process, presumably in all transfected cells (Buganimet al., 2012). Here a downstream requirement for other ERR familymembers, ERRα and ERRγ, together with their coactivators PGC-1α/β, thatdefine a distinct sub-population of cells with dramatically enhancedefficiency for iPSC generation was revealed. A transient surge in ERRα/γand PGC1α/β expression during reprogramming induced an early metabolicswitch epitomized by a transient OXPHOS burst and sustained enhancedglycolysis. These findings complement a recent study demonstratingstage-specific roles for HIF1α and HIF2α in the early increase inglycolytic metabolism (Mathieu et al., 2014, Haematologica 99,e112-114). The surprising functional divergence between ERRα/γ and ERRβadds a new dimension to the model for reprogramming, in which transientERRα/γ expression is important to drive an early hyper-energeticmetabolic state characterized by increased OXPHOS and glycolysis,whereas ERRβ is important for establishing induced pluripotency at laterreprogramming stages (Chen et al., 2008, Cell 133, 1106-1117; Martelloet al., 2012, Cell stem cell 11, 491-504; Zhang et al., 2008, TheJournal of biological chemistry 283, 35825-35833). The fact thatmetabolic reprogramming is a prerequisite of induced pluripotencyrevealed the functional relevance of a unique metabolic state toachieving cell plasticity. Furthermore, via cell sorting of Sca1/CD34double negative cells it was demonstrated that ERRγ and PGC-1β are earlymarkers of a newly defined sub-group of reprogramming progenitors. Insummary, these studies characterize a previously unrecognized, ERR/PGC-1dependent metabolic switch prior to establishment of inducedpluripotency in both human and mouse cells (FIG. 7G).

Example 5: ERRs Function Through IDH and α-Ketoglutarate to RegulateReprogramming

ERRα/γ regulate IDH gene expression and control the NAD+/NADH level inthe cells during reprogramming (FIG. 9A). As a key co-enzyme of histonedemethylase, α-ketoglutarate regulates the enzyme activity of severalhistone demethylases, such as KDM2 and KDM5, which act on H3K4Me2/3 andH3K9Me3. KDM stands for lysine (K) specific demethylase. As shown inFIG. 9A, ERRγ activates IDH3, which in turn catalyzes the oxidation ofisocitrate to α-ketoglutarate. During the reaction, NAD+, as electrondonor, is converted to NADH, thus decreasing the amount of NAD+ andincreasing the amount of NADH and decreasing the NAD+/NADH ratio(increasing NADH/NAD+ ratio)(FIG. 9B). Under the regulation ofα-ketoglutarate, histone demethylases demethylate histones at the lysinesite. For example, H3K4Me3 is demethylated to H3K4Me1. The demethylationof the histone leads to global changes in enhancer and promoterlandscape, and subsequently transcriptome dynamics.

IDH3 gene expression was upregulated during reprogramming of a cellpopulation (FIG. 9C). On day six of reprogramming, the relativeexpression levels of IDH3α, IDH3β, and IDH3γ genes were measured. Toevaluate the IDH3 gene expression in response to ERRα expression level,fibroblast cells were infected with a lentivirus expressing GFP underthe control of human ERRα promoter. GFP expression was used to markinfected cells and was subsequently used to FACS sort the cells intothose with high infection (ERRα−GFP+) and low infection (ERRα−GFP−).IDH3 α, β and γ gene expression was upregulated in cells expressing highlevels of ERRα (GFP+ cells) relative to corresponding control cells.Wild type (WT) fibroblasts, which are not infected, and cells with mockinfection (infected with vector only) serve as controls.

The α-ketoglutarate level in early reprogramming (day 5) depends on ERRγlevel in mouse reprogramming cells. In cells where ERRγ expression levelwas reduced through shRNA silencing, the relative abundance ofα-ketoglutarate was lower (FIG. 9D).

Inhibition of α-ketoglutarate-dependent histone demethylases led toreduced reprogramming efficiency (FIGS. 9E and 9F). Fewer iPS colonieswere formed after treatment of D-2-hydroxyglutarate (D-2-HG) orL-2-hydroxyglutarate (L-2-HG), which competitively inhibitα-ketoglutarate-dependent histone demethylases. Reprogramming efficiencywas significantly decreased after D-2-HG or L-2-HG treatment. L-2-HG isknown to be a more potent competitor than D-2-HG. Correspondingly,L-2-HG treatment led to more significant decrease of reprogramming(n=4-6, *P<0.05, *P<0.01) (FIGS. 9E and 9F). The determination of theabundance of α-ketoglutarate is well known to those skilled in the art.For example, commercial kits are available to quantify α-ketoglutarate.See, e.g.,http://www.biovision.com/alpha-ketoglutarate-colorimetric-fluorometric-assay-kit-2943.html,the content of which is incorporated by reference.

Example 6: ERRα Labels a Metabolically Active Subpopulation During EarlyReprogramming

During early reprogramming, ERRα expressing cells and ERRαnon-expressing cells were separated by GFP-based FACS analysis andRNA-seq was performed on each cell population (FIG. 10A). KEGG geneontology analysis was performed to identify the genes enriched in theERRα expressing population. The highly expressed genes in GFP+ cellswere associated with oxidative phosphorylation and other metabolicprocesses, which correlate with the known function of ERRα. The KEGGgene ontology analysis is well known to those skilled in the art. See,e.g., Mao et al., Automated genome annotation and pathway identificationusing the KEGG Orthology (KO) as a controlled vocabulary,Bioinformatics, 2005, 21(19): 3787-93, the content of which isincorporated by reference.

Example 7: The Promoter/Enhancer Landscapes are Different Between ERRα+and ERRα− Reprogramming Population

The promoter/enhancer landscapes were characterized in reprogrammingpopulations. In ERRα+ populations, H3 histone lysine 4 dimethylated(H3K4Me2) levels were decreased in the enhancer/promoter region of genesthat function in fibroblast identity, such as SNAI1 and ZEB2, comparedwith levels in cells that did not express detectable ERRα. This suggeststhat ERRα may be involved in the silencing of fibroblast specificepigenetic modifications.

The opposite changes were observed in genes that function inreprograming, such as Oct4 and Sox2. That is, the H3K4Me2 level wasincreased in the enhancer/promoter region of these genes, suggestingthat ERRα+ population contains cells whose pluripotency circuitry arepoised to be activated.

Methods for characterizing the promoter/enhancer landscape measurementis well known to those skilled in the art. One example is to useChromatin Immunoprecipitation assays (ChIP assays) to identify apolynucleotide associated with a histone with a modified amino acid,such as methylated lysine and quantify the level of the modification ofthe amino acid in a cell population. See, e.g., Chromatin Assembly andAnalysis, Current Protocols in Molecular Biology, Chapter 21 (Ausubel etal. eds., 2011), the content of which is incorporated by reference. Theexperiments described above were performed with the following methodsand materials.

Methods

Mouse embryonic fibroblasts (MEFs) were isolated from embryonic day (E)13.5 embryos obtained from wild-type and ERRγ-deficient mice (Alaynicket al., 2007). Retroviruses and lentiviruses were produced in HEK293Tcells, and 12 to 14 days after infection MEFs were fixed for staining.Reprogramming of MEFs and human lung fibroblast IMR90s was done aspreviously described (Kawamura et al., 2009, Nature 460, 1140-1144;Sugii et al., 2010, Proceedings of the National Academy of Sciences ofthe United States of America 107, 3558-3563; Takahashi et al., 2007,Cell 126, 663-676; Wei et al., 2013, Cell stem cell 2013 Jul. 3;13(1):36-47; Yu et al., 2007, Science, New York, N.Y., 318, 1917-1920).

Reprogramming

Mouse reprogramming was performed as previously described, withmodifications (Kawamura et al., 2009, Nature 460, 1140-1144; Sugii etal., 2010, Proceedings of the National Academy of Sciences of the UnitedStates of America 107, 3558-3563; Takahashi and Yamanaka, 2006, Cell126, 663-676; Yu et al., 2007, Science, New York, N.Y., 318, 1917-1920).For retroviral reprogramming, pMX-based retroviral vectors harboringeach of the mouse reprogramming genes (c-Myc, Klf4, Oct4, or Sox2;Addgene) were transfected along with gag/pol and VSV-G envelope genesinto HEK293T cells using Lipofectamine (Invitrogen). For lentivirusproduction, tet-inducible lentiviral vectors containing OSKM (Wei etal., 2009) were transfected together with pspax2 and pMD2.G (Addgene).Two days after transfection, supernatants containing viruses werecollected and filtered through a 0.45-m filter. For retroviralreprogramming, a total of 1×10⁴ (MEFs (passages 2-4) were infected withretroviral mixtures in 12-well plates (day 0). One well was used toquantify cell numbers for each group. Control cells were transduced withGFP retrovirus alone to determine infection efficiencies. On day 2,one-fifth of the cells were passaged onto gelatin-coated plates with MEFfeeder layers (Millipore) and cultured in Knockout (KO)-DMEM containingL-glutamine (2 mM), nucleosides (1×), NEAA (nonessential amino acid;1×), β-mercaptoethanol (1×), and LIF (1,000 units/mL), with 15% knockoutserum replacement (KSR, Millipore or Invitrogen). Media was changedevery other day. On days 7-10, cells were either immunostained forassessing efficiencies or derived into individual colonies fordownstream analyses.

For reprogramming of IMR90 fibroblasts, cells were infected with thecombination of human reprogramming retroviruses (c-Myc, Klf4, Oct4, orSox2 in pMXs; Addgene) that had been produced in 293T cellscotransfected with gag/pol and VSV-G as described above. EGFP retroviruswas included at 1/40 volume as internal controls for transductionefficiencies. One well from each group was reserved for quantifying cellnumbers. On day 2, cells were passaged onto 12-well plates containingMEF feeder cells (for generating iPSCs) or onto 6-cm dishes without MEF(for collecting mRNAs at day 5). Cells were cultured in Knockout(KO)-DMEM plus 20% knockout serum replacement (KSR) supplemented withβ-mercaptoethanol (0.1%), NEAA (1×), Glutamax (1%), and 10 ng/mL FGF2.Media was changed every day. Reprogramming of MEFs using an induciblelentiviral system was performed as previously described (Wei et al.,2009). Doxycyline-inducible MEFs were isolated fromGt(ROSA)26Sortm1(rtTA*M2)JaeCol1a1tm4(tetO-Pou5f1,-Sox2,-Klf4,-Myc)Jae/J mice (Jackson Labs) andreprogramming was performed as previously described (Carey et al.,2010). ERRγ-iKO mice were generated by crossing ERRγlox/lox (generouslyprovided by Johan Auwerx) and B6.Cg-Tg(CAG-cre/Esr1)5Amc/j (JacksonLabs, Cat. No. 004682) and ERRγ-iKO MEFs were isolated from EmbryonicDay 14.5 embryos. The ERRγ-iKO MEFs were reprogrammed using theinducible lentiviral system (Wei et al., 2009) and were treated by4-hydroxytamoxifen (4-OHT) at final concentration 50 nM fromreprogramming day 0 to day 2. All procedures involving hiPS/hES cellswere approved by the Embryonic Stem Cell Research Oversight Committee atthe Salk Institute.

Microarray Analysis

RNA was extracted from OSKM-induced MEFsat days 3, 4, 5, 6, 7 withshERRα and GFP-infected IMR90 cells at day 5 using RNEASY® (QIAGEN). RNAwas DNASE® (AMBION) treated, reverse transcribed to first-strand cDNAusing a SUPERSCRIPT® II kit (Invitrogen), and then treated with RNase.Global gene expression analysis was performed as described (Narkar etal., 2011, Cell Metab 13, 283-293.).

RNA-Seq Library Generation

Total RNA was isolated from cell pellets treated with RNALATER® usingthe RNA mini kit (Qiagen) and treated with DNASEI® (Qiagen) for 30 minat room temperature. Sequencing libraries were prepared from 100-500 ngtotal RNA using the TRUSEQ® RNA Sample Preparation Kit v2 (Illumina)according to the manufacturer's protocol. Briefly, mRNA was purified,fragmented, and used for first-, then second-strand cDNA synthesisfollowed by adenylation of 3′ ends. Samples were ligated to uniqueadapters and subjected to PCR amplification. Libraries were thenvalidated using the 2100 BIOANALYZER® (Agilent), normalized, and pooledfor sequencing. RNA-Seq libraries prepared from two biologicalreplicates for each experimental condition were sequenced on theIllumina HISEQ® 2000 using bar-coded multiplexing and a 100 bp readlength.

High-Throughput Sequencing and Analysis Image analysis and base callingwere performed with Illumina CASAVA®-1.8.2. This yielded a median of29.9M usable reads per sample. Short read sequences were mapped to aUCSC mm9 reference sequence using the RNA-sequence aligner STAR® (Dobinet al., 2013, Bioinformatics. 29(1):15-21). Known splice junctions frommm9 were supplied to the aligner and de novo junction discovery was alsopermitted. Differential gene expression analysis, statistical testingand annotation were performed using CUFFDIFF® 2 (Trapnell et al., 2013,Nat Biotechnol. 31(1):46-53). Transcript expression was calculated asgene-level relative abundance in fragments per kilobase of exon modelper million mapped fragments and employed correction for transcriptabundance bias (Roberts et al., 2011, Genome biology 12, R22). RNA-Seqresults for genes of interest were also explored visually using the UCSCGenome Browser.Gene Expression Analysis by qPCR

Samples were run in triplicate and expression was normalized to thelevels of the housekeeping controls Rplp0 (36b4) for human and mouse.Samples were analyzed by qPCR, using SYBR® Green dye (Invitrogen).Endogenous versus exogenous reprogramming gene expression was performedas previously reported (Yang et al., 2006, Cell 126, 801-810).Statistical comparisons were made using Student's t test. Error bars aremean±SEM.

Immunohistochemistry and Cell Staining

Cells grown on dishes were immunostained using the VectaStain ABC kitand IMMPACT® DAB substrate (Vector Lab) with rabbit anti-mouse Nanog(Calbiochem), anti-human Nanog (Abcam).

Bioenergetic Assay

Measurements were made with a SEAHORSE® XF instrument. Adherent cellswere seeded in 96-well SEAHORSE® cell culture microplates at 20,000 perwell 16 hours before measurement. Approximately 60 minutes prior to theassay, culture media was exchanged with a low-buffered DMEM assay mediawith 20 mM glucose and 1 mM sodium pyruvate. For measurement of maximaloxidative phosphorylation (OXPHOS) capacity, Oligomycin (finalconcentration 1.2 μM), Carbonyl cyanide-4

(trifluoromethoxy)phenylhydrazone (FCCP, final concentration 404),Antimycin A (final concentration 1 μM) and Rotenone (final concentration2 μM) were added per manufacturer's instruction. The oxygen consumptionrate (OCR) and extracellular acidification rate (ECAR) value werefurther normalized by measuring the cell number in each well usingHOECHST® 33342 staining followed by quantification of fluorescence at355 excitation and 460 emission. The baseline OCR was defined by theaverage value for the first 4 measurements. The maximal OXPHOS capacitywas defined by the difference between average OCR after addition ofCarbonyl cyanide-4 (trifluoromethoxy)phenylhydrazone (FCCP, minute88-120) and OCR after addition of antimycin A and rotenone (minute131-163).

shRNA Knockdown

shRNA constructs for mouse and human ERRα/γ and PGC-1α/β, as well ascontrol shRNA, were purchased from OPENBIOSYSTEMS®. Lentiviral shRNAwere produced in 293T cells and polybrene (6 μg/ml) was used intransduction. For reprogramming experiments, cells were transduced withlentiviral shRNA at day 0 of reprogramming.

Live Cell Staining, Alkaline Phosphatase Staining, and Cell Sorting

Cells were incubated with culture media containing FITC-conjugatedanti-Sca1 (1:50, Biolegend) and Phycoerythrin (PE)-conjugated anti-CD34(1:100, Biolegend) antibodies for 30 minutes, washed, then maintained inculture. Alkaline phosphatase staining was performed onformaldehyde-fixed cells using 4-Nitro blue tetrazolium chloride (450mg/ml) and 5-Bromo-4-chloro-3-indolyl phosphate (175 mg/ml) in NTMTsolution (0.1M NaCl, 0.1M Tris pH 9.5, 50 mM MgCl2, and 0.1% TWEEN®20).OSKM-infected cells were fluorescence-activated cell sorted (FACS,FACSAria, BD Biosciences) 5 days after infection using FITC-conjugatedanti-Sca1 (1:100) and phycoerythrin (PE)-conjugated anti-CD34 antibodies(1:200), and subsequently cultured for iPS cell formation.

In Vitro Differentiation

iPS cells were differentiated in vitro by embryoid body formation(Kawamura et al., 2009, Nature 460, 1140-1144) with some modification.Briefly, hanging droplets (1500 single cells at 60 cells/μl in mouse EScell media without LIF) were suspended on petri-dish lids for two orthree days prior to suspension culture. Six days after differentiation,embryoid bodies were plated on gelatinized dishes for 1-2 weeks. Geneexpression of pluripotency markers (Oct4, Sox2, Nanong, and E-Ras) andgerm-layer markers (AFP, Pdx1, and GATA6 for endoderm; GATA4, SMα-actin, and Cardiac α-actin for mesoderm; Cdx2, Pax6, and Mtap2 forectoderm) was determined by QPCR. Values were standardized to GAPDH andnormalized to undifferentiated mouse ES cells.

Blastocyst Injections for Chimeric Mice

Mouse iPS cells (derived from C57BL/6N MEFs) were injected into BALB/chost blastocysts and transferred into 2.5 dpc ICR pseudopregnantrecipient females. Chimerism was ascertained after birth by theappearance of black coat color (from iPS cell) in albino host pups.High-contribution chimeras were crossed to C57BL/6N mice to test forgermline transmission.

NAD+/NADH Assay Intracellular NAD+ and NADH levels were measured byNAD+/NADH Assay Kit (Abcam, San Francisco, Calif.) as per manufacturer'sinstructions. Briefly, 2×10⁵ cells were washed with cold PBS andextracted with NADH/NAD Extraction Buffer by two freeze/thaw cycles (20min on dry ice, then 10 min at room temperature). Total NAD (NADt) andNADH were detected in 96-well plates and color was developed and read at450 nm. NAD/NADH Ratio is calculated as: [NADt−NADH]/NADH.

Measurement of ATP

Intracellular ATP was measured by ATP assay kit (Sigma-Aldrich)according to manufacturer's directions. Briefly, 1×10⁴ cells were washedwith cold PBS and ATP extracted with ATP extraction buffer. Amounts ofATP were detected in 384-well plates and measured with a luminometer.

ChIP-Seq Library Construction, Sequencing and Data Analysis

ChIP-Seq libraries were constructed using standard Illumina protocols,validated using the 2100 BioAnalyzer (Agilent), normalized and pooledfor sequencing. Libraries were sequenced on the Illumina HiSeq 2500using barcoded multiplexing and a 50-bp read length. Short DNA readswere demultiplexed using Illumina CASAVA v1.8.2. Reads were alignedagainst the mouse mm9 using the Bowtie aligner allowing up to 2mismatches in the read. Only tags that map uniquely to the genome wereconsidered for further analysis. Subsequent peak calling and motifanalysis were conducted using HOMER, a software suite for ChIP-Seqanalysis. The methods for HOMER, which are described below, have beenimplemented and are freely available at http://biowhat.ucsd.edu/homer/.One tag from each unique position was considered to eliminate peaksresulting from clonal amplification of fragments during the ChIP-Seqprotocol. Peaks were identified by searching for clusters of tags withina sliding 200 bp window, requiring adjacent clusters to be at least 1 kbaway from each other. The threshold for the number of tags thatdetermine a valid peak was selected for a false discovery rate of <0.01,as empirically determined by repeating the peak finding procedure usingrandomized tag positions. Peaks are required to have at least 4-foldmore tags (normalized to total count) than input or IgG control samplesand 4-fold more tags relative to the local background region (10 kb) toavoid identifying regions with genomic duplications or non-localizedbinding. Peaks are annotated to gene products by identifying the nearestRefSeq transcriptional start site. Visualization of ChIP-Seq results wasachieved by uploading custom tracks onto the UCSC genome browser.

RNA-seq and data analysis Total RNA was isolated using Trizol(Invitrogen) and the RNeasy mini kit (Qiagen). RNA purity and integritywere confirmed using an Agilent Bioanalyzer. Libraries were preparedfrom 100 ng total RNA (TrueSeq v2, Illumina) and singled-endedsequencing performed on the Illumina HiSeq 2500, using bar-codedmultiplexing and a 100 bp read length, yielding a median of 34.1M readsper sample. Read alignment and junction finding was accomplished usingSTAR and differential gene expression with Cuffdiff 2 utilizing UCSC mm9as the reference sequence.

Chromatin Immunoprecipitation

Cells were then harvested for ChIP assay. Briefly, after fixation,nuclei were isolated, lysed and sheared with a Diagenode Bioruptor toyield DNA fragment sizes of 200-1000 base pairs followed byimmunoprecipitation using H3K4Me2 antibodies (Abcam ab32356).

ChIP-Seq Data Analysis

The procedure was as previously described (Barish et al., 2010; Ding etal., 2013). Briefly, short DNA reads were demultiplexed using IlluminaCASAVA v1.8.2. Reads were aligned against the human hg18 (NCBI Build36.1) using the Bowtie aligner allowing up to 2 mismatches in the read.Only tags that map uniquely to the genome were considered for furtheranalysis. Subsequent peak calling and motif analysis were conductedusing HOMER, a software suite for ChIP-Seq analysis. The methods forHOMER, which are described below, have been implemented and are freelyavailable at http://biowhat.ucsd.edu/homer/. One tag from each uniqueposition was considered to eliminate peaks resulting from clonalamplification of fragments during the ChIP-Seq protocol. Peaks wereidentified by searching for clusters of tags within a sliding 200 bpwindow, requiring adjacent clusters to be at least 1 kb away from eachother. The threshold for the number of tags that determine a valid peakwas selected for a false discovery rate of <0.01, as empiricallydetermined by repeating the peak finding procedure using randomized tagpositions. Peaks are required to have at least 4-fold more tags(normalized to total count) than input or IgG control samples and 4-foldmore tags relative to the local background region (10 kb) to avoididentifying regions with genomic duplications or non-localized binding.Peaks are annotated to gene products by identifying the nearest RefSeqtranscriptional start site. Visualization of ChIP-Seq results wasachieved by uploading custom tracks onto the UCSC genome browser.

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1.-45. (canceled)
 46. An in vitro method of generating a mammalianinduced pluripotent stem cell progenitor or induced pluripotent stemcell, the method comprising: (a) introducing into a population ofmammalian cells polynucleotides encoding reprogramming markers Oct4,Sox2, Klf4 and cMyc on day 1, which markers are expressed in the cell;(b) culturing a subpopulation of cells within the population ofmammalian cells which comprises cells having increased expression ofendogenous estrogen related receptor alpha (ERRα) or estrogen relatedreceptor gamma (ERRγ) and of at least one of peroxisomeproliferator-activated receptor gamma co-activator 1 alpha (PGC-1α),peroxisome proliferator-activated receptor gamma co-activator 1 beta(PGC-1β), or isocitrate dehydrogenase 3 (IDH3) by at least day 3 to day5 following step (a); wherein the cells further display an increasedmetabolic rate defined by an increase in one or both of extracellularacidification rate and oxygen consumption rate and enhanced glycolysis,relative to cells which do not express the reprogramming markers, ERRα,or ERRγ; and, optionally, (c) isolating a cell from the culturedsubpopulation of cells following step (b), thereby generating amammalian induced pluripotent stem cell progenitor or inducedpluripotent stem cell in vitro.
 47. The method of claim 46, wherein themammalian cells are human cells and ERRα expression is increased by atleast day 5 following step (a).
 48. The method of claim 47, wherein thehuman cells are selected from the group consisting of fibroblasts, lungfibroblasts, adipocytes, and IMR90 cells.
 49. The method of claim 46,wherein the mammalian cells are non-human mammalian cells and ERRγexpression is increased by at least day 3 following step (a).
 50. Themethod of claim 49, wherein the non-human mammalian cells are selectedfrom the group consisting of fibroblasts, embryonic fibroblasts, lungfibroblasts, and adipose stem cells.
 51. The method of claim 46,wherein, in step (a), the polynucleotides encoding the Oct4, Sox2, Klf4and cMyc reprogramming markers are introduced in one or more retroviralvectors.
 52. The method of claim 46, wherein the subpopulation of cellsexpresses increased levels of one or more of nicotinamide adeninedinucleotide (NADH), α-ketoglutarate, cellular ATP, NADH/NAD+ ratio, ATPsynthase in mitochondria (ATP5G1), succinate dehydrogenase (SDHB),isocitrate dehydrogenase (IDH3) and NADH dehydrogenase (NDUFA2),superoxide dismutase 2 (SOD2), NADPH oxidase 4 (NOX4) and catalase (CAT)following expression of the Oct4, Sox2, Klf4 and cMyc reprogrammingmarkers compared with cells not expressing said reprogramming markers.53. An in vitro method of reprogramming a population of human somaticcells to produce induced pluripotent stem cells (iPSCs), the methodcomprising: (i) transducing the population of human somatic cells with avector encoding the reprogramming markers Oct 4, Sox2, Klf4, and c-Mycon day 1, wherein the reprogramming markers are expressed in the humansomatic cells; (ii) culturing a subpopulation of cells within thepopulation of human somatic cells following step (i), wherein thesubpopulation of cells have increased expression of endogenous estrogenrelated receptor alpha (ERRα) and peroxisome proliferator-activatedreceptor gamma co-activator 1 (PGC-1) by at least day 5 following step(i); wherein oxidative phosphorylation is induced in the cells toachieve an oxidative burst and enhanced glycolysis following theexpression of ERRα in the cells; thereby reprogramming the cellsfollowing steps (i) and (ii) to produce iPSCs.
 54. The method of claim53, wherein the population of human somatic cells comprises cellsselected from the group consisting of fibroblasts, adipocytes, and IMR90cells.
 55. The method of claim 53, wherein expression of reactive oxygenspecies of superoxide dismutase 2 (SOD2), NADPH oxidase 4 (NOX4) andcatalase (CAT) are upregulated in the subpopulation of cells at days 5-8following step (i).
 56. The method of claim 53, wherein energymetabolism in the cells of step (ii) is increased by inducing expressionof at least one of ATP synthase in mitochondria (ATP5G1), succinatedehydrogenase (SDHB), isocitrate dehydrogenase 3A (IDH3A) and NADHdehydrogenase (NDUFA2).
 57. The method of claim 53, wherein the cellsare transduced with one or more lentivirus vectors comprisingpolynucleotides encoding the reprogramming markers.
 58. An in vitromethod of reprogramming a population of non-human mammalian somaticcells to produce induced pluripotent stem cells (iPSCs), the methodcomprising: (i) transducing the population of non-human mammaliansomatic cells with a vector encoding the reprogramming markers Oct 4,Sox2, Klf4, and c-Myc on day 1, wherein the reprogramming markers areexpressed in the non-human mammalian somatic cells; and (ii) culturing asubpopulation of cells within the population of non-human mammaliansomatic cells following step (i), wherein the subpopulation of cellshave increased expression of endogenous estrogen related receptor gamma(ERRγ) and peroxisome proliferator-activated receptor gamma co-activator1 (PGC-1) by at least day 3 following step (i); wherein oxidativephosphorylation is induced in the cells to achieve an oxidative burstand enhanced glycolysis following the expression of ERRγ in the cells;thereby reprogramming the cells following steps (i) and (ii) to produceiPSCs.
 59. The method of claim 58, wherein the population of non-humanmammalian somatic cells comprises fibroblast cells or embryonicfibroblast cells.
 60. The method of claim 58, wherein expression ofreactive oxygen species of superoxide dismutase 2 (SOD2), NADPH oxidase4 (NOX4) and catalase (CAT) is upregulated in the subpopulation of cellsat days 5-8 following step (i).
 61. The method of claim 58, whereinenergy metabolism in the cells of step (ii) is increased by inducingexpression of at least one of ATP synthase in mitochondria (ATP5G1),succinate dehydrogenase (SDHB), isocitrate dehydrogenase 3A (IDH3A) andNADH dehydrogenase (NDUFA2).
 62. A cellular composition comprising aneffective amount of a mammalian induced pluripotent stem cell progenitorof claim 46, or a cellular descendant thereof, and a pharmaceuticallyacceptable diluent, excipient, or carrier.
 63. The cellular compositionof claim 62, wherein the mammalian induced pluripotent stem cellprogenitor can give rise to a pancreatic cell, a neuronal cell, or acardiac cell.